EGFR-binding modular recognition domains

ABSTRACT

EGFR-binding modular recognition domains (MRDs) and complexes containing such MRDs linked to antibodies are described. The use of such MRDs and complexes are also described.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates generally to compositions containing multivalent multispecific complexes and to compositions containing multivalent and monovalent multispecific complexes having scaffolds, such as antibodies, that support such binding functionalities. The invention also generally relates to methods of making these multispecific compositions and the diagnostic and therapeutic uses of these compositions.

Background

In recent years, drug discovery efforts have primarily focused on identifying agents that modulate preselected individual targets. However, agents directed to individual targets frequently show limited efficacies and poor safety and resistance profiles, as a result of the robustness, redundancy, crosstalk, compensatory signaling networks and anti- or counter-signaling network activities associated with the therapeutic target. Consequently, drug discovery efforts have increasingly been directed toward the discovery of new multicomponent based therapies.

The development of bispecific or multi-specific molecules that target two or more targets simultaneously offers a novel and promising solution for discovering new systems-oriented multitargeted agents demonstrating improved efficacy and pharmacological properties over conventional monotherapies. Numerous attempts to develop multispecific molecules have been based on immunoglobulin-like domains or subdomains. For example, traditionally, bispecific antibodies have been prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Other immunoglobulin-like domain-based technologies that have created multispecific, and/or multivalent molecules include dAbs, diabodies, TandAbs, nanobodies, BiTEs, SMIPs, DNLs, Affibodies, Fynomers, Kunitz Domains, Albu-dabs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knobs-in-Holes, DuoBodies™ and triomAbs. Although each of these molecules may bind one or more targets, they each present challenges with respect to retention of typical Ig function (e.g., half-life, effector function), production (e.g., yield, purity), valency, simultaneous target recognition, and bioavailability.

Other attempts to generate multispecific and multivalent molecules have relied on alternative scaffolds, based VASP polypeptides, Avian pancreatic polypeptide (aPP), Tetranectin (based on CTLD3), Affilin (based on γB-crystallin/ubiquitin), knottins, SH3 domains, PDZ domains, Tendamistat, Transferrin, an ankyrin consensus repeat domain (e.g., DARPins), lipocalin protein folds (e.g., Duocalins), fibronectin (see for example, US Application Publ. Nos. 2003/0170753 and 20090155275 which are herein incorporated by reference), a domain of protein A (e.g., Affibodies), thioredoxin. Other attempts have relied on alternative scaffolds fuse or associate polypeptides of interest with albumin (e.g., ALBUdAb (Domantis/GSK) and ALB-Kunitz (Dyax)), unstructured repeat sequences of 3 or 6 amino acids (e.g., PASylation® technology and XTEN® technology), and sequences containing elastin-like repeat domains (see for example, U.S. Pat. Appl. No. 61/442,106, which is herein incorporated by reference). To date, these technologies have demonstrated limited clinical potential as robust platforms for developing diverse multispecific and multivalent therapeutic compositions.

The genetic complexity of most human malignancies and other disorders strongly suggest that interfering with a single target or pathway associated with these disorders is unlikely to produce optimal or sustained therapeutic benefit. There is, therefore, a great need for developing multispecific and multivalent therapeutics such as multispecific antibodies that are capable of interfering with the activity of multiple targets and/or signaling mechanisms in or to optimize the therapeutic benefits of treatments directed towards these disorders.

BRIEF SUMMARY OF THE INVENTION

The invention relates to compositions containing multivalent as well as multivalent and monovalent, multispecific complexes having scaffolds, such as antibodies, that support such binding functionalities. The invention is based in part on the surprising discovery that multispecific and multivalent binding compositions, such as those generated using the ZYBODY™ platform (Zyngenia, Inc.; see, e.g., Intl. Pub. No. WO 2009/088805 which is herein incorporated by reference) demonstrate dramatic synergistic biological activity compared to conventional monotherapy combinations. This synergistic activity is expected to extend to novel therapies, for treating or preventing cancer, diseases or disorders of the immune system (e.g., autoimmune diseases such as, rheumatoid arthritis, and IBD), skeletal system (e.g., osteoporosis), cardiovascular system (e.g., stroke, heart disease), nervous system (e.g., Alzheimer's), infectious disease (e.g., HIV), and other diseases or disorders described herein or otherwise known in the art.

In one embodiment, the invention is directed to treating a disease or disorder by administering a therapeutically effective amount of a multivalent and monovalent multispecific composition to a patient in need thereof. In a further embodiment, the invention is directed to treating a disease or disorder by administering a therapeutically effective amount of a multivalent and multispecific MRD-containing antibody to a patient in need thereof.

In one embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for three or more targets. In an additional embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for four or more targets. In another embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for five or more targets. According to some embodiments, at least 1, 2, 3, 4 or more of the targets are located on a cell surface. According to some embodiments, at least 1, 2, 3, 4 or more of the targets are soluble targets (e.g., chemokines, cytokines, and growth factors). In additional embodiments, the multivalent and monovalent multispecific composition binds 1, 2, 3, 4 or more of the targets described herein.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with cancer. In a further embodiment the targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4 or more different signaling pathways or modes of action associated with cancer.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In a further embodiment the targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4 or more different signaling pathways or modes of action associated with a disease or disorder of the immune system.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease. In a further embodiment the targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4, 5 or more different signaling pathways or modes of action associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease. In a further embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4, 5 or more of the targets described herein.

In one embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for three or more targets. In an additional embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for four or more targets. In an additional embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for five or more targets.

In one embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for three or more targets. In an additional embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for four or more targets. In another embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for five or more targets. According to some embodiments, at least 1, 2, 3, 4, or more of the targets are associated with the cell membrane. According to some embodiments, at least 1, 2, 3, 4, or more of the targets are soluble targets (e.g., chemokines, cytokines, and growth factors). In additional embodiments, the multivalent and monovalent multispecific composition binds 1, 2, 3, 4, or more of the targets described herein.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with cancer. In a further embodiment the targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4, or more different signaling pathways or modes of action associated with cancer.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In a further embodiment the targets bound by the—multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4, or more different signaling pathways or modes of action associated with a disease or disorder of the immune system.

In additional embodiments, the multivalent and monovalent multispecific composition binds (1) a target on a cell or tissue of interest (e.g., a tumor associated antigen on a tumor cell, an immune cell, a diseased cell or an infectious agent) and (2) a target on an effector cell. According to one embodiment, the binding of one or more targets by the multivalent and monovalent multispecific composition directs an immune response to a cell, tissue, infectious agent, or other location of interest in a patient. In some embodiments the effector cell is a leukocyte, such as a T cell or natural killer cell. In other embodiments, the effector cell is an accessory cell, such as a myeloid cell or a dendritic cell.

In additional embodiments, the multivalent and monovalent multispecific composition binds (1) a target on a cell or tissue of interest (e.g., a tumor associated antigen on a tumor cell, an immune cell, a diseased cell or an infectious agent) and (2) a target on a leukocyte, such as a T-cell receptor molecule. According to one embodiment, the binding of one or more targets by the multivalent and monovalent multispecific composition directs an immune response to an infectious agent, cell, tissue, or other location of interest in a patient. For example, in some embodiments the multivalent and monovalent multispecific composition binds a target on the surface of a T cell. In particular embodiments, the composition binds a CD3 target selected from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, TCR alpha, TCR beta, and multimers of proteins in the CD3 (TCR) complex. In specific embodiments the multivalent and monovalent multispecific composition binds CD3. In other embodiments, the multivalent and monovalent multispecific composition binds CD2. In additional embodiments, the multivalent and monovalent multispecific composition binds a target expressed on a natural killer cell. Thus, in some embodiments, the multivalent and monovalent multispecific composition binds a target selected from: CD2, CD56, and CD161.

In additional embodiments, the multivalent and monovalent multispecific composition binds a target expressed on an accessory (e.g., myeloid) cell. In some embodiments, the multivalent and monovalent multispecific composition binds a target selected from: CD64 (i.e., Fc gamma RI), an MHC class 2 and its invariant chain, TLR1, TLR2, TLR4, TLR5, and TLR6.

In further embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD containing antibody) has a single binding site (i.e., is monovalent) for a target. In some embodiments, the multivalent and monovalent multispecific composition has a single binding site for a target on a leukocyte, such as a T-cell (e.g., CD3), and multiple binding sites (i.e., is multivalent) for a target on a cell or tissue of interest (e.g., a tumor associated antigen on a tumor cell, such as a target disclosed herein). In further embodiments, the multispecific composition contains single binding sites for 2 different targets (i.e., monovalently binds more than one different target). In particular embodiments, the cell or tissue of interested is a cancer cell, immune cell, diseased cell, or an infectious agent.

In some embodiments, a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has a single binding site for CD3. In further embodiments, the multivalent and monovalent multispecific composition has a single binding site for CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen or other target disclosed herein). In additional embodiments, the multispecific composition has a single binding site for CD3 and a single binding site for a different target (i.e., monovalently binds CD3 and a different target). In other embodiments, a multivalent and monovalent multispecific composition has a single binding site for CD3 epsilon. In further embodiments, the multivalent and monovalent multispecific composition has a single binding site for CD3 epsilon and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen or other target disclosed herein). In further embodiments, the multispecific composition has a single binding site for CD3 epsilon and a single binding site for a different target (i.e., monovalently binds CD3 epsilon and a different target). In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for a target on a cancer cell selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer.

In further embodiments, the invention is directed to treating a disease or disorder by administering a therapeutically effective amount of a multivalent and monovalent multispecific composition that has a single binding site for a target (i.e., that monovalently binds a target) to a patient in need thereof. In some embodiments, the administered multivalent and monovalent multispecific composition has a single binding site for a target on a leukocyte such as a T-cell (e.g., CD3). In further embodiments, the administered multivalent and monovalent multispecific composition has a single binding site for a target on a leukocyte such as a T-cell (e.g., CD3) and multiple binding sites for (i.e., is capable of multivalently binding) a target located on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell). In further embodiments, the multispecific composition has a single binding site for a target on a leukocyte (e.g., CD3) and a single binding site for a different target. In some embodiments, the cell of interest is a tumor cell from a cancer selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer. In additional embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for a target on a neurological tumor. In particular embodiments, the neurological tumor is a glioma (e.g., a glioblastoma, glioblastoma multiforme (GSM), and astrocytoma), ependymoma, oligodendroglioma, neurofibroma, sarcoma, medulloblastoma, primitive neuroectodermal tumor, pituitary adenoma, neuroblastoma or cancer of the meninges (e.g., meningioma, meningiosarcoma and gliomatosis).

In further embodiments, the invention is directed to treating a disease or disorder by administering to a patient in need thereof, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that has a single binding site for a target (i.e., that monovalently binds a target) and multiple binding sites for 1, 2, 3, 4, 5 or more different targets. In further embodiments, the multivalent and monovalent multispecific composition has single binding sites for 2 different targets. In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for a target on a cancer cell selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer.

In additional embodiments, the invention is directed to treating a disease or disorder by administering to a patient in need thereof, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that has a single binding site for CD3 (e.g., CD3 epsilon) that monovalently binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets located on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell). In some embodiments, the administered multivalent and monovalent multispecific composition has a single binding site for CD3 (e.g., CD3 epsilon) and a single binding site for a different target and also has multiple binding sites for a target located on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell). In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for a target on a cancer cell selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer.

In further embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has a single binding site for (i.e., monovalently binds) a cell surface target that requires multimerization for signaling. In some embodiments, the multivalent and monovalent multispecific composition has a single binding site for a growth factor receptor. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for a TNF receptor superfamily member. In additional embodiments, the multispecific composition additionally has a single binding site for a different target (i.e., monovalently binds more than one different target).

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds a target associated with an endogenous blood brain barrier (BBB) receptor mediated transport system and is capable of crossing to the brain (cerebrospinal fluid) side of the BBB. In some embodiments, the multivalent and monovalent multispecific composition has two or more binding sites for a target antigen associated with an endogenous BBB receptor mediated transport system. In additional embodiments, the multivalent and monovalent multispecific composition has a single binding site for a target associated with an endogenous BBB receptor mediated transport system (e.g., the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor mediated transport systems). In further embodiments, the multivalent and monovalent multispecific composition additionally binds 1, 2, 3, 4, 5, or more targets located on the brain side of the BBB. In particular embodiments, the MRD-containing antibody binds 1, 2, 3, 4, 5, or more targets associated with a neurological disease or disorder. In another embodiment, the multivalent and monovalent multispecific composition is administered to a patient to treat a brain cancer, metastatic cancer of the brain, or primary cancer of the brain. In a further embodiment, the multivalent and monovalent multispecific composition is administered to a patient to treat brain injury, stroke, spinal cord injury, or to manage pain.

In additional embodiments, targets bound by the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease. In a further embodiment a targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4, 5 or more different signaling pathways or modes of action associated with one or more of the above diseases or disorders. In a further embodiment, the multivalent and monovalent multispecific composition binds 1, 2, 3, 4, 5 or more of the targets described herein.

In one embodiment, the multivalent and monovalent multispecific composition is a ZYBODY™ (referred to herein as an “MRD-containing antibody,” or the like). In a further embodiment, the MRD-containing antibody contains binding sites for three or more targets. In an additional embodiment, the MRD-containing antibody contains 2 binding sites for four or more targets. In an additional embodiment, the MRD-containing antibody contains 2 binding sites for five or more targets.

In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains 2 binding sites for three or more targets. In an additional embodiment, the multispecific composition (e.g., MRD-containing antibody) contains 2 binding sites for four or more targets. In another embodiment, the multispecific composition (e.g., MRD-containing antibody) contains 2 binding sites for five or more targets. According to some embodiments, at least 1, 2, 3, 4 or more of the targets are located on a cell surface. According to some embodiments, at least 1, 2, 3, 4 or more of the targets are soluble targets (e.g., chemokines, cytokines, and growth factors). In additional embodiments, the MRD-containing antibody binds at least 1, 2, 3, 4, 5 or more of the targets described herein.

In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) are associated with cancer. In a further embodiment the targets bound by MRD-containing antibody are associated with 1, 2, 3, 4 or more different signaling pathways or modes of action associated with cancer.

In additional embodiments, a target bound by the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is associated with a disease or disorder of the immune system. In a further embodiment the targets bound by the MRD-containing antibody are associated with 1, 2, 3, 4, 5 or more different signaling pathways or modes of action associated with a disease or disorder of the immune system.

In additional embodiments, a target bound by the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is associated with a disease or disorder of the skeletal system, cardiovascular system, nervous system, or an infectious disease. In a further embodiment a target bound by the MRD-containing antibody is associated with 1, 2, 3, 4 or more different signaling pathways or modes of action associated with one or more of the above diseases or disorders. In another embodiment, the MRD-containing antibody binds 1, 2, 3, 4 or more of the targets described herein.

The multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies) provide the ability to selectively target multiple targets (e.g., receptors and microenvironment associated targets) having for example, different, overlapping, or redundant mechanisms of action associated with the etiology or pathophysiology of a disease or disorder.

In additional embodiments, the invention encompasses a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that is covalently or otherwise associated with a cytotoxic agent. According to some embodiments, the cytoxic agent is covalently attached to an MRD-containing antibody by a linker. According to some embodiments, the cytotoxic agent is a chemotherapeutic agent, growth inhibitory agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotope (i.e., a radioconjugate), or prodrug. The compositions of the invention are optionally linked to the cytotoxic agent by a linker. In particular embodiments, a linker attaching the multivalent and monovalent multispecific composition and the cytotoxic agent is cleavable by a protease. In particular embodiments, a linker attaching the multivalent and monovalent multispecific composition and the cytotoxic agent is cleavable under low pH or reducing conditions. Methods of using composition-cytoxic agent compositions of the invention (e.g., MRD-containing antibody drug conjugates) are also encompassed by the invention.

In additional embodiments, the multivalent and multispecific compositions is covalently or otherwise associated with a cytotoxic agent selected from, for example, a toxin, enterotoxin, neurotoxin, leukocidin or hemolysin, a chemotherapeutic agent, a drug moiety (e.g., a chemotherapeutic agent or prodrug), an antibiotic, a radioactive isotope, a chelating ligand (e.g., DOTA, DOTP, DOTMA, DTPA and TETA), and a nucleolytic enzyme. In particular embodiments, the cytotoxic agent is selected from auristatin and dolostantin, MMAE, MMAF, and a maytansinoid derivative (e.g., the DM1 (N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine), DM3 (N(2′)-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine), and DM4 (N(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine). In additional embodiments, the multivalent and multispecific composition is covalently or otherwise associated with a cytostatic agent.

In further embodiments, a multivalent and monovalent multispecific composition of the invention (e.g., an MRD-containing antibody) is administered in combination with a multitargeting therapeutic. In one embodiment, a multivalent and monovalent multispecific composition is administered in combination with a multitargeting protein kinase inhibitor. In another embodiment, a multivalent and monovalent multispecific composition is administered in combination with an NFKB inhibitor. In an additional embodiment, a multivalent and monovalent multispecific composition is administered in combination with an HDAC inhibitor. In a further embodiment, a multivalent and monovalent multispecific composition is administered in combination with an HSP70 or HSP90 inhibitor. In a further embodiment, a multivalent and monovalent multispecific composition is administered in combination with chemotherapy.

In some embodiments, a multivalent and monovalent multispecific composition of the invention (e.g., an MRD-containing antibody) is administered in combination with a monospecific therapeutic (e.g., a monoclonal antibody).

In some embodiments, a multivalent and monovalent multispecific composition of the invention is a full-length antibody comprising at least one modular recognition domain (MRD). In some embodiments, the full-length antibody comprises multiple MRDs. In additional embodiments, the full-length antibody comprises more than one type of MRD (i.e., multiple MRDs having the same or different specificities). Also embodied in the present invention are variants and derivatives of such antibody complexes.

The MRDs of the MRD containing antibodies can be operably attached to the antibodies at any location on the antibody (e.g., the amino terminus of the heavy chain or light chain or the carboxyl terminus of the heavy chain or light chain), can be linked at the same or different termini, and are optionally operably linked to one another or to the antibody by a linker.

The antibodies of the MRD containing antibodies can be any immunoglobulin molecule that binds to an antigen and can be of any type, class, or subclass. In some embodiments, the antibody is humanized or human. In other embodiments, the antibodies also include modifications that do not interfere with their ability to bind antigen. In particular embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) include modifications that increase ADCC, decrease ADCC, increase CDC, or decrease CDC, that increase antibody half-life, or decrease antibody half-life compared to the antibody without the modification.

The antibodies of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention can be any antibody that binds to a target of therapeutic or diagnostic value. In preferred embodiments, the antibody of the MRD-containing antibody binds to a validated target. In some embodiments, the antibodies corresponding to the MRD containing antibodies are in clinical trials for regulatory approval. In some embodiments, the antibodies corresponding to the MRD containing antibodies are marketed.

In one embodiment, the antibody binds to a cell surface antigen. In another embodiment, the antibody binds to an angiogenic factor. In a further embodiment, the antibody binds to an angiogenic receptor.

In some embodiments, the antibody of the MRD-containing antibody binds to a target selected from: EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-I receptor, VEGF, VEGF-R and prostate specific membrane antigen. In additional embodiments the antibody of the MRD-containing antibody binds to VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, FGFR2, and CD20.

In one embodiment, the antibody of the MRD-containing antibody binds to EGFR. In another specific embodiment, the antibody is Erbitux®, nimotuzumab, or zalutumumab (e.g., Genmab). In another embodiment, the antibody binds to the same epitope as Erbitux® antibody or competitively inhibits binding of the Erbitux® antibody to EGFR. In a further specific embodiment, the antibody is the Erbitux® antibody. In one specific embodiment, the antibody binds to the same epitope as Erbitux®, nimotuzumab, zalutumumab (e.g., Genmab) antibody. In another specific embodiment, the antibody component, MRD component, and/or MRD-containing antibody competitively inhibits binding of Erbitux®, nimotuzumab, zalutumumab antibody to EGFR.

In one embodiment, an MRD-containing antibody binds EGFR and a target selected from: HGF, CD64, CDCP1, RON, cMET, ErbB2, ErbB3, IGF1R, PLGF, RGMa, PDGFRa, PDGFRb, VEGFR1, VEGFR2, TNFRSF10A (DR4), TNFRSF10B (DR5), TNFRSF21 (DR6), IGF1,2, IGF2, CD3, CD4, and NKG2D. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) binds at least 1, 2, 3, 4, 5 or more of these targets. In specific embodiments, the antibody component of the MRD-containing antibody binds EGFR. In further embodiments, the antibody component of the MRD-containing antibody is nimotuzumab, zalutumumab. In specific embodiments, the antibody component of the MRD-containing antibody is Erbitux®.

In a specific embodiment, the antibody of the MRD-containing antibody binds to ErbB2. In one embodiment, the antibody is HERCEPTIN® (trastuzumab) antibody or competitively inhibits HERCEPTIN® (trastuzumab) antibody binding to ErbB2.

In another specific embodiment, the antibody binds to VEGF. In another specific embodiment, the antibody binds to the same epitope as AVASTIN® (bevacizumab) antibody or competitively inhibits AVASTIN® antibody. In a further specific embodiment, the antibody is the AVASTIN® antibody.

In some embodiments, the antibody binds to a target that is associated with a disease or disorder of the immune system. In one embodiment, the antibody binds to TNF. In another specific embodiment, the antibody binds to the same epitope as HUMIRA® (adalimumab) antibody or competitively inhibits HUMIRA® antibody. In a further specific embodiment, the antibody is the HUMIRA® antibody. In one embodiment, the antibody binds to TNF. In another specific embodiment, the antibody binds to the same epitope as SIMPONI™ (golimumab) antibody or competitively inhibits SIMPONI™ antibody. In a further specific embodiment, the antibody is the SIMPONI™ antibody.

In some embodiments, the antibody component of the MRD containing antibody binds to a target that is associated with a disease or disorder of the metabolic, cardiovascular, musculoskeletal, neurological, or skeletal system. In other embodiments, the antibody component of the MRD containing antibody binds to a target that is associated with yeast, fungal, viral or bacterial infection or disease.

The invention also encompasses, MRDs, compositions comprising MRDs (e.g., 0antibodies and/or other compositions), polynucleotides encoding MRDs, methods of manufacturing MRDs, and methods of using the MRDs of the invention. In one embodiment, the MRD is about 2 to 150 amino acids. In another embodiment, the MRD is about 2 to 60 amino acids. MRDs can be linked to an antibody or other MRDs directly or through a linker. The MRDs can be any target binding peptide. In some embodiments, the MRD target is a soluble factor. In other embodiments, the MRD target is a transmembrane protein such as a cell surface receptor. In another embodiment, the target of the MRD is a cellular antigen. In a specific embodiment, the target of the MRD is CD20.

In another embodiment, the target of the MRD is an integrin. In one aspect, the peptide sequence of the integrin targeting MRD is YCRGDCT (SEQ ID NO:3). In another aspect, the peptide sequence of the integrin targeting MRD is PCRGDCL (SEQ ID NO:4). In yet another aspect, the peptide sequence of the integrin targeting MRD is TCRGDCY (SEQ ID NO:5). In another aspect, the peptide sequence of the integrin targeting MRD is LCRGDCF (SEQ ID NO:6).

In an additional embodiment, the target of the MRD is an angiogenic cytokine. In one aspect, the peptide sequence of the angiogenic cytokine targeting (i.e., binding) MRD is MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7).

In one embodiment, the target of the MRD is ErbB2. In another embodiment, the target to which the MRD binds is ErbB3. In an additional embodiment, the target to which the MRD binds is tumor-associated surface antigen or an epithelial cell adhesion molecule (Ep-CAM).

In one embodiment, the target to which the MRD binds is VEGF. In one aspect, the peptide sequence of the VEGF targeting MRD is VEPNCDIHVMWEWECFERL (SEQ ID NO:13).

In one embodiment, the target to which the MRD binds is an insulin-like growth factor-I receptor (IGF1R). An illustrative IGF1R targeting MRD includes, for example, a peptide sequence having the formula: NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37);

In one embodiment, the target of the MRD is a tumor antigen. The “tumor antigen” as used herein may be understood as both those antigens (including mutations) exclusively expressed on tumor cells (i.e., tumor-specific antigens) and those antigens expressed on tumor cells and normal cells (e.g., antigens overexpressed on tumor cells).

In one embodiment, the target of the MRD is an epidermal growth factor receptor (EGFR). In another embodiment of the present invention, the target of the MRD is an angiogenic factor. In an additional embodiment, the target of the MRD is an angiogenic receptor.

In another embodiment, the MRD is a vascular homing peptide.

In one embodiment, the target of the MRD is a nerve growth factor.

In another embodiment, the antibody and/or MRD binds to EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-I receptor, or prostate specific membrane antigen.

The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence encoding an MRD-containing antibody. In one aspect, a vector comprises a polynucleotide sequence encoding an MRD-containing antibody. In another aspect, the polynucleotide sequence encoding an MRD-containing antibody is operatively linked with a regulatory sequence that controls expression on the polynucleotide. In an additional aspect, a host cell comprises the polynucleotide sequence encoding an MRD-containing antibody.

Methods of making multivalent and multispecific compositions (e.g., MRD-containing antibodies) are also provided, as are the use of these MRD-antibody fusions in diagnostic and therapeutic applications. The present invention also relates to methods of designing and making multivalent and multispecific compositions (e.g., MRD-containing antibodies) having a full-length antibody comprising a MRD. In one aspect, the MRD is derived from a phage display library. In another aspect, the MRD is derived from natural ligands. In another aspect, the MRD is derived from yeast display or RNA display technology.

The present invention also relates to a method of treating or preventing a disease or disorder in a subject (patient) in need thereof, comprising administering an antibody comprising an MRD to the subject (patient). In one aspect, the disease is cancer. In another aspect, undesired angiogenesis in inhibited. In another aspect, angiogenesis is modulated. In yet another aspect, tumor growth is inhibited.

Certain embodiments provide for methods of treating or preventing a disease, disorder, or injury comprising administering to a patient in need thereof, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) to a patient in need thereof. In some embodiments, the disease, disorder or injury is cancer. In other embodiments, the disease, disorder or injury is a disorder of the immune system. In one embodiment, the disorder of the immune system is inflammation. In another embodiment, the disorder of the immune system is an autoimmune disease. In an additional embodiment, the disorder of the immune system is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, systemic lupus erythematous, inflammatory bowel disease, psoriasis, diabetes, ulcerative colitis, and multiple sclerosis. In one embodiment, the disease, disorder or injury is a metabolic disease. In another embodiment, the disease, disorder, or injury is an infectious disease. In specific embodiments, the infectious disease is human immunodeficiency virus (HIV) infection or AIDS, botulism, anthrax, or clostridium difficile. In other embodiments, the disease, disorder, or injury is neurological. In a specific embodiment, the neurological disease, disorder or injury is pain. In a more specific embodiment, the pain is, acute pain or chronic pain.

In another embodiment, a method of treatment or prevention comprising administering an additional therapeutic agent along with an antibody comprising an MRD is provided. In other embodiments, the methods of treatment or prevention comprise administering an antibody comprising more than one type of MRD.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the schematic representation of different designs of multi-specific and multivalent molecules. MRDs are depicted as triangles, circles, diamonds, and squares.

FIG. 2A shows a typical peptibody as a C-terminal fusion with the heavy chain of Fc.

FIG. 2B shows an MRD containing antibody with a C-terminal MRD fusion with the light chain of the antibody.

FIG. 2C shows an MRD containing antibody with an N-terminal MRD fusion with the light chain of the antibody.

FIG. 2D shows an MRD containing antibody with unique MRD peptides fused to each terminus of the antibody.

FIG. 3 depicts the results of an enzyme linked immunosorbent assay (ELISA) in which integrin and Ang2 were bound by an anti-integrin antibody (JC7U) fused to an Ang2 targeting MRD (2×Con4).

FIG. 4 depicts the results of an ELISA in which integrin and Ang2 were bound by an anti-integrin antibody (JC7U) fused to an Ang2 targeting MRD (2×Con4).

FIG. 5 depicts the results of an ELISA in which an anti-ErbB2 antibody was fused to an MRD which targets Ang2.

FIG. 6 depicts the results of an ELISA in which an Ang2 targeting MRD was fused to a hepatocyte growth factor receptor (cMET) binding antibody.

FIG. 7 depicts the results of an ELISA in which an integrin targeting MRD was fused to an ErbB2-binding antibody.

FIG. 8 depicts the results of an ELISA in which an integrin targeting MRD was fused to a hepatocyte growth factor receptor binding antibody.

FIG. 9 depicts the results of an ELISA in which an insulin-like growth factor-I receptor targeting MRD was fused to an ErbB2-binding antibody.

FIG. 10 depicts the results of an ELISA in which a VEGF-targeting MRD was fused to an ErbB2-binding antibody.

FIG. 11 depicts the results of an ELISA in which an integrin targeting MRD was fused to a catalytic antibody.

FIG. 12 depicts the results of an ELISA in which an Ang2-targeting MRD was fused to a catalytic antibody.

FIG. 13 depicts the results of an ELISA in which an integrin targeting MRD and an Ang2 targeting MRD were fused to an ErbB2-binding antibody.

FIG. 14 depicts the results of an ELISA in which an integrin targeting MRD was fused to an ErbB2-binding antibody.

FIG. 15 depicts the results of an ELISA in which an integrin, Ang2, or insulin-like growth factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptor-binding antibody with a short linker peptide.

FIG. 16 depicts the results of an ELISA in which an integrin, Ang2, or insulin-like growth factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptor-binding antibody with a long linker peptide.

FIG. 17A depicts the dose response curves of MRD-maltose binding protein (MBP) fusions assayed for direct binding to Ang2.

FIG. 17B indicates MRD-MBP fusion proteins tested, the amino acid sequence of the MRD, and the EC50 values (calculated using a 4 parameter fit). The MXD sequence motif in the MRD components of the MRD-MBP fusions is underlined and mutated residues are in bold and italics.

FIG. 18A depicts the results of an assay for direct binding of a HERCEPTIN® based zybody (i.e. an MRD containing HERCEPTIN® antibody sequences) antibody-MRDs and a HERCEPTIN® antibody to Her2 (ErbB2) Fc in the presence of biotinylated Ang2. Binding was detected with HRP-conjugated anti-human kappa chain mAb.

FIG. 18B depicts the results of an assay for direct binding of a HERCEPTIN® based zybody (i.e., an MRD containing HERCEPTIN® antibody sequences) and a HERCEPTIN® antibody to Her2 Fc in the presence of biotinylated Ang2. Binding was detected with horseradish peroxidase (HRP)-conjugated streptavidin.

FIG. 19A depicts the results of an assay for direct binding of antibody-MRDs and an AVASTIN® antibody to VEGF in the presence of biotinylated Ang2. Binding was detected with HRP-conjugated anti-human kappa chain mAb.

FIG. 19B depicts the results of an assay for direct binding of antibody-MRDs and an AVASTIN® antibody to VEGF in the presence of biotinylated Ang2. Binding was detected with HRP-conjugated streptavidin.

FIG. 20A depicts the results of a flow cytometry assay which demonstrates that antibody-MRDs simultaneously bind Her2 and Ang2 on BT-474 breast cancer cells.

FIG. 20B depicts binding of antibody-MRDs to HER2 on BT-474 breast cancer cells.

FIG. 21 depicts the results of an ELISA assay that demonstrates the inhibitory effect of antibody-MRDs on TIE-2 binding to plate immobilized Ang2.

FIG. 22 depicts the results of a competitive binding assay that demonstrates the inhibition of binding of biotinylated antibody by antibody-MRD and unlabeled antibody.

FIG. 23 depicts the results of a competitive binding assay that illustrates the inhibition of labeled antibody binding to BT-474 cells by antibody-MRDs and unlabeled antibody.

FIG. 24A depicts the fitted dose curves illustrating the inhibition of BT-474 cell proliferation by HERCEPTIN® with the lm32 MRD (SEQ ID NO:8) fused to the heavy chain and HERCEPTIN®.

FIG. 24B depicts the fitted dose curves illustrating the inhibition of BT-474 cell proliferation by HERCEPTIN® with the lm32 MRD fused to the light chain and HERCEPTIN®.

FIG. 24C depicts the fitted dose curves illustrating the inhibition of BT-474 cell proliferation by HERCEPTIN® with the 2×con4 MRD fused to the heavy chain and HERCEPTIN®.

FIG. 25A depicts the results of a cytotoxicity assay illustrating ADCC-mediated killing of BT-474 cells by HERCEPTIN® with the lm32 MRD fused to the heavy chain, HERCEPTIN® with the lm32 MRD fused to the light chain, and HERCEPTIN®.

FIG. 25B depicts the results of a cytotoxicity assay illustrating ADCC-mediated killing of BT-474 cells by HERCEPTIN® with the 2×con4 MRD fused to the heavy chain, and HERCEPTIN®.

FIG. 26A depicts the inhibition of HUVEC proliferation by AVASTIN® with the lm32 MRD fused to the heavy chain and AVASTIN® using HUVECs obtained from GlycoTech (Gaithersburg, Md.).

FIG. 26B depicts the inhibition of HUVEC proliferation by AVASTIN® with the lm32 MRD fused to the heavy chain and AVASTIN® using HUVECs obtained from Lonza.

FIG. 27 depicts the effect of RITUXIMAB®, HERCEPTIN®, and an MRD-containing antibody on tumor volume in vivo.

FIG. 28 depicts the increased effect of an antibody-containing MRD on receptor phosphorylation and AKT activation compared to the effect of an antibody in combination with the MRD.

FIG. 29A depicts the increased effect of a bispecific MRD-containing antibody on cell proliferation compared to the effect of the antibody or the antibody in combination with the MRD.

FIG. 29B depicts the increased effect of a pentaspecific MRD-containing antibody on cell proliferation compared to the effect of the antibody or the antibody in combination with the MRD.

FIG. 30 depicts the increased efficacy of a HUMIRA antibody containing an Ang2-binding MRD in an arthritis model compared to HUMIRA.

FIG. 31 shows inhibition of EGF-induced signaling in SK-BR3 cells by zybodies.

FIG. 32 shows inhibition of Heregulin-induced signaling in SK-BR3 zybodies.

FIG. 33 shows inhibition of EGF and Heregulin-induced signaling in SK-BR3 cells by zybodies.

FIG. 34 shows a bar-graph (A) and flow-cytometry results (B) depicting the down-regulation of EGFR expression on SK-BR3 cells by zybodies.

FIG. 35 shows down-regulation of EGFR in SKBR3 cells by zybodies.

FIG. 36 shows the cytotoxic effects of MRD-containing antibodies expressing different valencies of a DR5 agonizing MRD on three different mAb scaffolds using a cell viability assay. FIG. 36A, demonstrates that a tetravalent trastuzumab scaffold-based zybody demonstrates higher cytotoxic activity compared to corresponding bivalent zybodies. FIGS. 36B-C demonstrate that octavalent trastuzumab and palivizumab antibody scaffold-based zybodies containing eight copies of the DR5 MRD, demonstrate higher cytotoxic activity compared to their respective corresponding tetravalent zybodies. FIG. 36D and demonstrates that the multi-epitopic targeting of non-DR5 receptors (her2 and EGFR, respectively) enhances the cytotoxic activity of a tetra-valent DR5 targeting MRD. FIG. 36F demonstrates that the mab scaffold of the zybody (e.g., cetuximab (CET), trastuzumab (TRA), and palivizumab (PAL)) can influence the apparent cytotoxic potency of DR5 targeting MRDs.

FIG. 37 demonstrates that Ang-binding peptides bind may differ in the relative binding to Ang1 and Ang4. The assayed peptides are ANG100 (SEQ ID NO:621), ANG126 (SEQ ID NO:232); ANG129 (SEQ ID NO:234); ANG156 (SEQ ID NO:255); ANG157 (SEQ ID NO:256); ANG163 (SEQ ID NO:261); ANG179 (SEQ ID NO:270); ANG200 (SEQ ID NO:628); ANG303 (SEQ ID NO:331); ANG318 (SEQ ID NO:625); ANG335 (SEQ ID NO:332); and ANG599 (SEQ ID NO:367).

DETAILED DESCRIPTION OF THE INVENTION

The following provides a description of multivalent and monovalent multispecific compositions (e.g., MRD-containing antibodies containing at least one modular recognition domain (MRD)). The linkage of one or more MRDs to an antibody results in a multi-specific molecule of the invention that retains structural and functional properties of traditional antibodies or Fc optimized antibodies and can readily be synthesized using conventional antibody expression systems and techniques. The antibody can be any suitable antigen-binding immunoglobulin, and the MRDs can be any suitable target-binding peptide. The MRDs can be operably linked to any location on the antibody, and the attachment can be direct or indirect (e.g., through a chemical or polypeptide linker). Compositions of antibodies comprising an MRD, methods of manufacturing antibodies comprising an MRD, and methods of using antibodies comprising MRDs are also described in the sections below.

The invention also encompasses MRDs, compositions comprising MRDs, polynucleotides encoding MRDs, methods of manufacturing MRDs, and methods of using the MRDs of the invention.

The section headings used herein are for organizational purposes only and are not to be construed as in any way limiting the subject matter described.

Standard techniques may be used for recombinant DNA molecule, protein, and antibody production, as well as for tissue culture and cell transformation. Enzymatic reactions and purification techniques are typically performed according to the manufacturer's specifications or as commonly accomplished in the art using conventional procedures such as those set forth in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) and Sambrook et al., (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) (both herein incorporated by reference), or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, are those known and used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

I. Definitions

The terms “multivalent and monovalent multispecific complexes”, “multivalent and multispecific complexes”, “MRD-containing antibodies,” “antibody-MRD molecules,” “MRD-antibody molecules,” “antibodies comprising an MRD” and “Zybodies” are used interchangeably herein and do not encompass a peptibody. Each of these terms may also be used herein to refer to a “complex” of the invention. Multivalent and monovalent multispecific complexes can contain MRDs, antibodies, cytoxic agents, and binding motifs in addition to MRDs that bind to one or more targets. For example, a multivalent and monovalent multispecific complex (e.g., an MRD-containing antibody) can contain a portion of, or a derivative of, a binding sequence contained in antibody (e.g., a single binding domain, a ScFv, a CDR region) and/or can also include a cytotoxic agent (e.g., a therapeutic agent). Such molecules are also described in U.S. Provisional Application No. 61/481,063, which is herein incorporated by reference in its entirety. The terms “multivalent and monovalent multispecific complex(es)” and “multivalent and monovalent multispecific complexes” as used herein therefore refer to compositions that are able to bind 2 or more targets and that contain one binding site and/or multiple binding sites for different epitopes. Thus, this term is intended to include complexes containing multiple binding sites for each different epitope bound by the complex, or alternatively, complexes that contain at least one single binding site for a different epitope. The different epitopes can be on the same or different targets. The targets of the multivalent and multi-specific complexes can be on the same or different cells. Multivalent and monovalent multispecific complexes can be multivalent and multispecific and can therefore bind two or more targets and have two or more binding sites for each of the targets bound by the complex. Multivalent and monovalent multispecific complexes can also have one (or more) single binding sites for one (or more) target(s) and multiple binding sites for other targets and accordingly, these complexes are monovalent (with respect to the single binding site(s)), multivalent and multispecific. Moreover, multivalent and monovalent multispecific complexes can be monovalent and multispecific and thus, only contain single binding sites for two or more different targets.

The term “multivalent and monovalent multispecific complex-drug complex” or “MRD-containing antibody-cytotoxic agent” as used herein, refers to a multivalent and monovalent multispecific complex containing one or more cytotoxic agents.

The term “cytotoxic agent” as used herein, includes any agent that is detrimental to cells including for example, substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include a chemotherapeutic agent, a drug moiety (e.g., a cytokine or prodrug), an antibiotic, a radioactive isotope, a chelating ligand (e.g., DOTA, DOTP, DOTMA, DTPA and TETA), a nucleolytic enzyme, a toxins such as a small molecule toxin or enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants of these toxins. In particular embodiments, the cytotoxic agent is a member selected from: auristatin, dolostantin, MMAE, MMAF, a maytansinoid derivative (e.g., the DM1 (N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine), DM3 (N(2′)-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine) and DM4 (N(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).

The term “antibody” is used herein to refer to immunoglobulin molecules that are able to bind antigens through an antigen binding domain (i.e., antibody combining site). The term “antibody” includes polyclonal, oligoclonal (mixtures of antibodies), and monoclonal antibodies, chimeric, single chain, and humanized antibodies. The term “antibody” also includes human antibodies. In some embodiments, an antibody comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In other embodiments, the antibody is a homomeric heavy chain antibody (e.g., camelid antibodies) which lacks the first constant region domain (CH1) but retains an otherwise intact heavy chain and is able to bind antigens through an antigen binding domain. The variable regions of the heavy and light chains in the antibody-MRD fusions of the invention contain a functional binding domain that interacts with an antigen.

The term “monoclonal antibody” typically refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. As used herein, a “monoclonal antibody” may also contain an antibody molecule having a plurality of antibody combining sites (i.e., a plurality of variable domains), each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody. Thus, as used herein, a “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of one or two (in the case of a bispecific monoclonal antibody) antigenic determinants, or epitopes. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, yeast, and transgenic animals.

A “dual-specific antibody” is used herein to refer to an immunoglobulin molecule that contains dual-variable-domain immunoglobulins, where the dual-variable-domain can be engineered from any two monoclonal antibodies.

The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity and/or affinity while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

The term “humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity and/or affinity (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity and/or affinity. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539, U.S. Pat. No. 4,816,567, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494, WO86/01533; WO8702671; Boulianne et al., Nature 312:643 (1984); and Neuberger et al., Nature 314:268 (1985), each of which is herein incorporated by reference in its entirety.

As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin or one or more human germlines and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al., A human antibody may still be considered “human” even if amino acid substitutions are made in the antibody. Examples of methods used to generate human antibodies are described in: Int. Appl. Publ. Nos. WO98/24893, WO92/01047, WO96/34096, and WO96/33735; European Pat. No. 0 598 877; U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598; and Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), which are herein incorporated by reference.

An “antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term “immunoreact” in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain (i.e., antibody combining site) formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which are herein incorporated by reference). “Humanized antibody” or “chimeric antibody” includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The terms “T lymphocyte,” “T cell,” “T cells,” and “T cell population,” are used interchangeably herein to refer to a cell or cells which display on their surface one or more antigens characteristic of T cells, for example, CD3 and CD11b. The term includes progeny of a T cell or T cell population. A “T lymphocyte” or “T cell” includes a cell which expresses CD3 on its cell surface and a T cell antigen receptor (TCR) capable of recognizing antigen when displayed on the surface of autologous cells, or any antigen-presenting matrix, together with one or more MHC molecules or, one or more non-classical MHC molecules. The term “T cells” may refer to any T cells, including for example, lymphocytes that are phenotypically CD3⁺ i.e., express CD3 on the cell surface.

As used herein, CD3, is used to refer individually or collectively to a molecule expressed as part of the T cell receptor and having a meaning as typically ascribed to it in the art. In humans, the term CD3 encompasses all known CD3 subunits, for example CD3 delta, CD3 epsilon, CD3 gamma, and CD3 zeta (TCR zeta), as well as CD3 alpha (TCR alpha), and CD3 beta (TCR beta) in individual or independently combined form.

The term “peptibody” refers to a peptide or polypeptide which comprises less than a complete, intact antibody. A peptibody can be an antibody Fc domain attached to at least one peptide. A peptibody does not include antibody variable regions, an antibody combining site, CH1 domains, or Ig light chain constant region domains.

The term “naturally occurring” when used in connection with biological materials such as a nucleic acid molecules, polypeptides, host cells, and the like refers to those which are found in nature and not modified by a human being.

The term “domain” as used herein refers to a part of a molecule or structure that shares common physical or chemical features, for example hydrophobic, polar, globular, helical domains or properties, e.g., a protein binding domain, a DNA binding domain or an ATP binding domain. Domains can be identified by their homology to conserved structural or functional motifs.

A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s) to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions and non-conservative substitutions which do not eliminate polypeptide or antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

A “modular recognition domain” (MRD) or “target binding peptide” is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to a target molecule. The amino acid sequence of a MRD can typically tolerate some degree of variability and still retain a degree of capacity to bind the target molecule. Furthermore, changes in the sequence can result in changes in the binding specificity and in the binding constant between a preselected target molecule and the binding site. In one embodiment, the MRD is an agonist of the target it binds. An MRD agonist refers to a MRD that in some way increases or enhances the biological activity of the MRD's target protein or has biological activity comparable to a known agonist of the MRD's target protein. In another embodiment, the MRD is an antagonist of the target it binds. An MRD antagonist refers to an MRD that blocks or in some way interferes with the biological activity of the MRD's target protein or has biological activity comparable to a known antagonist or inhibitor of the MRD's target protein.

“Cell surface receptor” refers to molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell. An example of a cell surface receptor of the present invention is an activated integrin receptor, for example, an activated αvβ3 integrin receptor on a metastatic cell. As used herein, “cell surface receptor” also includes a molecule expressed on a cell surface that is capable of being bound by an MRD containing antibody of the invention.

As used herein, a “target binding site” or “target site” is any known, or yet to be defined, amino acid sequence having the ability to selectively bind a preselected agent. Exemplary reference target sites are derived from the RGD-dependent integrin ligands, namely fibronectin, fibrinogen, vitronectin, von Willebrand factor and the like, from cellular receptors such as ErbB2, VEGF, vascular homing peptide or angiogenic cytokines, from protein hormones receptors such as insulin-like growth factor-I receptor, epidermal growth factor receptor and the like, and from tumor antigens.

The term “epitope” or “antigenic determinant” are used interchangeably herein and refer to that portion of any molecule capable of being recognized and specifically bound by a particular binding agent (e.g., an antibody or an MRD). When the recognized molecule is a polypeptide, epitopes can be formed from contiguous amino acids and noncontiguous amino acids and/or other chemically active surface groups of molecules (such as carbohydrates) juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

An antibody, MRD, antibody-containing MRD, or other molecule is said to “competitively inhibit” binding of a reference molecule to a given epitope if it binds to that epitope to the extent that it blocks, to some degree, binding of the reference molecule to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. As used herein, an antibody, MRD, antibody-containing MRD, or other molecule may be said to competitively inhibit binding of the reference molecule to a given epitope, for example, by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

The term “protein” is defined as a biological polymer comprising units derived from amino acids linked via peptide bonds; a protein can be composed of two or more chains.

A “fusion polypeptide” is a polypeptide comprised of at least two polypeptides and optionally a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature. The two polypeptides may be operably attached directly by a peptide bond or may be linked indirectly through a linker described herein or otherwise known in the art.

The term “operably linked,” as used herein, indicates that two molecules are attached so as to each retain functional activity. Two molecules are “operably linked” whether they are attached directly (e.g., a fusion protein) or indirectly (e.g., via a linker).

The term “linker” refers to a peptide located between the antibody and the MRD or between two MRDs. Linkers can have from about 1 to 20 amino acids, about 2 to 20 amino acids, or about 4 to 15 amino acids. One or more of these amino acids may be glycosylated, as is well understood by those in the art. In one embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In another embodiment, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine Thus, in some embodiments, the linker is selected from polyglycines (such as (Gly)₅, and (Gly)₈), poly(Gly-Ala), and polyalanines. The linker can also be a non-peptide linker such as an alkyl linker, or a PEG linker. For example, alkyl linkers such as —NH—(CH₂)s-C(O)—, wherein s=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In certain embodiments, the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa. The peptide linkers may be altered to form derivatives. In some embodiments, the linker is a non-peptide linker such as an alkyl linker, or a PEG linker. In further embodiments, the linker is a “cleavable linker” facilitating release of an MRD or cytotoxic agent within a cell or in the proximity of the cell.

“Target cell” refers to any cell in a subject (e.g., a human or animal) that can be targeted by a multispecific and multivalent composition (e.g., an antibody-containing MRD) or MRD of the invention. The target cell can be a cell expressing or overexpressing the target binding site, such as an activated integrin receptor.

The term “immune response” refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells, or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, cosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils). Some effector cells express specific Fc receptors and carry out specific immune functions. In certain embodiments, an effector cell is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes and macrophages, which express FcR, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen or target cell. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of Fc alpha RI has been found to be up-regulated by G-CSF or GM-CSF. This enhanced expression increases the effector function of Fc alpha RI-bearing cells against targets. Exemplary functions of an effector cell include the phagocytosing or lysing of a target antigen or a target cell.

“Target cell” refers to any cell or pathogen whose elimination would be beneficial in a patient (e.g., a human or animal) and that can be targeted by a composition (e.g., antibody) of the invention.

“Patient,” “subject,” “animal” or “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens, amphibians, and reptiles. In some embodiments, the patient is a human.

“Treating” or “treatment” includes the administration of the antibody comprising an MRD of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, condition, or disorder, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, or disorder. Treatment can be with the antibody-MRD composition alone, the MRD alone, or in combination of either with one or more additional therapeutic agents.

As used herein, the terms “pharmaceutically acceptable,” or “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of therapeutically prohibitive undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

“Modulate” or “modulates” means adjustment or regulation of amplitude, frequency, degree, or activity. In another related aspect, such modulation may be positively modulated (e.g., an increase in frequency, degree, or activity) or negatively modulated (e.g., a decrease in frequency, degree, or activity).

“Cancer,” “tumor,” or “malignancy” are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize) as well as any of a number of characteristic structural and/or molecular features. A “cancerous tumor,” or “malignant cell” is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis. Examples of cancers that may be treated using the antibody-MRD fusions of the invention include solid tumors and hematologic cancers. Additional, examples of cancers that may be treated using the antibody-MRD fusions of the invention include breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g., leukemia), and prostate cancer. Further examples of cancer that may be treated using the multivalent and multispecific compositions (e.g., MRD-containing antibodies) include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers. Other types of cancer and tumors that may be treated using multivalent and multispecific compositions (e.g., MRD-containing antibodies) are described herein or otherwise known in the art.

An “effective amount” of an antibody, MRD, or MRD-containing antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose such as to bring about an observable change in the level of one or more biological activities related to the target to which the antibody, MRD, or MRD-containing antibody binds. In certain embodiments, the change increases the level of target activity. In other embodiments, the change decreases the level of target activity. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of an antibody, MRD, MRD-containing antibody, other multivalent and multispecific drug of the invention, or other drug effective to “treat” a disease or disorder in a patient or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce angiogenesis and neovascularization; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent or stop) tumor metastasis; inhibit, to some extent, tumor growth or tumor incidence; stimulate immune responses against cancer cells and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of “treating”. A “therapeutically effective amount” also may refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a composition of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic composition are outweighed by the therapeutically beneficial effects.

To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects (patients) prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Where embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the disclosed and/or claimed invention.

II. Modular Recognition Domains (MRDs)

The present invention describes an approach based on the adaptation of target binding peptides or modular recognition domains (MRDs) as fusions to catalytic or non-catalytic antibodies.

In certain embodiments, where the antibody component of the MRD-antibody fusion is a catalytic antibody, the MRD-antibody fusions provide for effective targeting to tumor cells or soluble molecules while leaving the prodrug activation capability of the catalytic antibody intact. MRDs can also extend the binding capacity of non-catalytic antibodies providing for an effective approach to extend the binding functionality of antibodies, particularly for therapeutic purposes.

One aspect of the present invention relates to development of a full-length antibody comprising at least one modular recognition domain (MRD). In another non-exclusive embodiment, the full-length antibody comprises more than one MRD, wherein the MRDs have the same or different specificities. In addition, a single MRD may be comprised of a tandem repeat of the same or different amino acid sequence that can allow for the binding of a single MRD to multiple targets and/or to a repeating epitope on a given target.

The interaction between a protein ligand and its target receptor site often takes place at a relatively large interface. However, only a few key residues at the interface contribute to most of the binding. The MRDs can mimic ligand binding. In certain embodiments, the MRD can mimic the biological activity of a ligand (an agonist MRD) or through competitive binding inhibit the bioactivity of the ligand (an antagonist MRD). MRDs in multivalent and multispecific compositions (e.g., MRD-containing antibodies) can also affect targets in other ways, e.g., by neutralizing, blocking, stabilizing, aggregating, or crosslinking the MRD target.

It is contemplated that MRDs of the present invention will generally contain a peptide sequence that binds to target sites of interests and have a length of about 2 to 150 amino acids, about 2 to 125 amino acids, about 2 to 100 amino acids, about 2 to 90 amino acids, about 2 to 80 amino acids, about 2 to 70 amino acids, about 2 to 60 amino acids, about 2 to 50 amino acids, about 2 to 40 amino acids, about 2 to 30 amino acids, or about 2 to 20 amino acids. It is also contemplated that MRDs have a length of about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 90 amino acids, about 10 to 80 amino acids, about 10 to 70 amino acids, about 10 to 60 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, or about 10 to 20 amino acids. It is further contemplated that MRDs have a length of about 20 to 150 amino acids, about 20 to 125 amino acids, about 20 to 100 amino acids, about 20 to 90 amino acids, about 20 to 80 amino acids, about 20 to 70 amino acids, about 20 to 60 amino acids, about 20 to 50 amino acids, about 20 to 40 amino acids, or about 20 to 30 amino acids. In certain embodiments, the MRDs have a length of about 2 to 60 amino acids. In other embodiments, the MRDs have a length of about 10 to 60 amino acids. In other embodiments, the MRDs have a length of about 10 to 50 amino acids. In additional embodiments, the MRDs have a length of about 10 to 40 amino acids. In additional embodiments, the MRDs have a length of about 10 to 30 amino acids.

In some embodiments, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd of less than 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. In one embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻⁵ M. In another embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻⁸ M. In another embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻⁹ M. In another embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻¹⁰ M. In another embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻¹¹ M. In another embodiment, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd less than 5×10⁻¹² M.

In specific embodiments, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind their targets with an off rate (k_(off)) of less than 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³ sec⁻¹. More preferably, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind their targets with an off rate (k_(off)) of less than 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹, or 10⁻⁷ sec⁻¹.

In other specific embodiments, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind their targets with an on rate (k_(on)) of greater than 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹, or 5×10⁴ M⁻¹ sec⁻¹. More preferably, one or more of the MRD components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind their targets with an on rate (k_(on)) of greater than 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹, or 10⁷ M⁻¹ sec⁻¹.

In some embodiments, the MRDs are affibodies. Affibodies represent a class of affinity proteins based on a 58-amino acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. This three helix bundle domain has been used as a scaffold for the construction of combinatorial phagemid libraries, from which affibody variants that bind a desired target molecule, such as one or more of the targets disclosed herein, can routinely be selected using phage display technology (see, e.g., Nord et al., Nat. Biotechnol. 15:772-7 (1997), and Ronmark et al., A, Eur. J. Biochem. 2002; 269:2647-55). Further details of Affibodies and methods of production thereof are provided by reference to U.S. Pat. No. 5,831,012, which is herein incorporated by reference in its entirety.

In other embodiments, an MRD of the invention (e.g., an MRD on an MRD-containing antibody) contains one or more amino acid residues or sequences of amino acid residues (including derivatives, analogs, and mimetics thereof) that are preferentially targeted by chemistries or other processes that covalently or non-covalently link a molecular entity to the MRD, as compared to, the MRD without the preferentially targeted sequences or the antibody component of the MRD-containing antibody. For example, in some embodiments, the amino acid sequence of the MRD contains one or more residues having a reactive side chain (e.g., cysteine or lysine) that allows for selective or preferential linkage of the MRD to cytotoxic agents (e.g., drug and prodrug conjugates, toxins, and bioactive ligands) or imaging agents.

The use of these “linking” MRDs to arm an MRD-comprising antibody with a “payload” overcomes many of the issues associated with antibody destabilization and reduction in antibody activity that have frequently been observed using conventional methods for generating immunotoxins. The “payload” component of an MRD-comprising antibody complex of the invention can be any composition that confers a beneficial therapeutic, diagnostic, or prognostic effect, or that provide an advantage in manufacturing, purifying or formulating an MRD-containing antibody. In some embodiments, the payload is a chemotherapeutic drug, or a prodrug, such as, doxorubicin or a maytansinoid-like drug. In additional embodiments, the payload is another MRD, a toxin, a chemotherapeutic drug, a catalytic enzyme, a prodrug, a radioactive nuclide, a chelator (e.g., for the attachment of lanthanides) or another component of the multivalent and multispecific compositions of the invention as described herein.

In nonexclusive embodiments, the MRD does not contain an antigen binding domain, or another antibody domain such as a constant region, a variable region, a complementarity determining region (CDR), a framework region, an Fc domain, or a hinge region. In one non-exclusive embodiment, the MRD does not contain an antigen binding domain. In another non-exclusive embodiment, the MRD does not contain three CDRs. In another non-exclusive embodiment, the MRD does not contain CDR1 and CDR2. In yet another non-exclusive embodiment, the MRD does not contain CDR1. In one nonexclusive embodiment, the MRD is not derived from a natural cellular ligand. In another nonexclusive embodiment, the MRD is not a radioisotope. In another nonexclusive embodiment, the MRD is not a protein expression marker such as glutathione S-transferase (GST), His-tag, Flag, hemagglutinin (HA), MYC or a fluorescent protein (e.g., GFP or RFP). In another nonexclusive embodiment, the MRD does not bind serum albumin. In an additional nonexclusive embodiment, the MRD is not a small molecule that is a cytotoxin. It yet another nonexclusive embodiment, the MRD does not have enzymatic activity. In another non-exclusive embodiment, the MRD has a therapeutic effect when administered alone and/or when fused to an Fc in a patient or animal model. In another non-exclusive embodiment, the MRD has a therapeutic effect when repeatedly administered alone and/or when fused to an Fc in a patient or animal model (e.g., 3 or more times over the course of at least six months).

In some embodiments, the MRD is conformationally constrained. In other embodiments, the MRD is not conformationally constrained. In some embodiments, the MRD contains one cysteine residue. The cysteine residue in the MRD can form an interchain bond (e.g., between cysteines within the same MRD, different peptide linked MRDs, and an MRD and a peptide linked immunoglobulin). In some embodiments, the MRD(s) participating in the interchain bond is/are associated with a single core target-binding domain. In other embodiments, the MRD(s) participating in the interchain bond is/are associated with multiple core target-binding domains. In an alternative embodiment, the cysteine residue in the MRD can form an interchain bond (e.g., between cysteines of non-peptide linked MRDs or an MRD and an immunoglobulin that are not linked by a peptide bind). In some embodiments, the MRD(s) associated with the interchain bond is/are associated with a single core target-binding domain (i.e., 2 MRDs located on different polypeptide chains form one or more interchain bonds and collectively form one target binding site). Thus, for example, the invention encompasses MRD-containing antibodies wherein MRDs located on the carboxyl terminus of the heavy chain interact (e.g., via disulfide bond) so as to form a single target binding site. In other embodiments, the MRD(s) associated with the interchain bond is/are associated with multiple core target-binding domains. Alternatively, as discussed herein, the MRD can contain one or more cysteine residues (or other residue having a reactive side chain (e.g., lysine)) that allows for selective or preferential linkage of the MRD to a cytotoxic agent.

In some embodiments, the MRD contains two cysteine residues outside the core target-binding domain. In some embodiments, the MRD contains two cysteine residues located within the core target-binding domain at each end of the target-binding domain. In some embodiments, a first cysteine is located near the terminus of the molecule (i.e. at the C-terminus of an MRD on the C-terminus of a linker or antibody chain or at the N-terminus of an MRD on the N-terminus of a linker or antibody chain). Thus, in some embodiments, a first cysteine is located within one amino acid, within two amino acids, within three amino acids, within four amino acids, within five amino acids, or within six amino acids of the terminus of the molecule. In some embodiments, a second cysteine is located near the MRD fusion location (i.e. at the N-terminus of an MRD on the C-terminus of a linker or antibody chain or at the C-terminus of an MRD on the N-terminus of a linker or antibody chain). Thus, in some embodiments, a second cysteine is located within one amino acid, within two amino acids, within three amino acids, within four amino acids, within five amino acids, within 10 amino acids, or within 15 amino acids from the MRD fusion.

In some embodiments, the MRD is capped with stable residues. In some embodiments, the MRD is disulfide capped. In some embodiments, the MRD does not contain cleavage sites.

In some embodiments, the MRD has been selected to not contain known potential human T-cell epitopes.

In some particular embodiments, the MRD has a particular hydrophobicity. For example, the hydrophobicity of MRDs can be compared on the basis of retention times determined using hydrophobic interaction chromatography or reverse phase liquid chromatography.

The MRD target can be any molecule that it is desirable for an MRD-containing antibody to interact with. For example, the MRD target can be a soluble factor or a transmembrane protein, such as a cell surface receptor. The MRD target can also be an extracellular component or an intracellular component. In certain non-exclusive embodiments, the MRD target is a factor that regulates cell proliferation, differentiation, or survival. In other nonexclusive embodiments, the MRD target is a cytokine. In another nonexclusive embodiment, the MRD target is a factor that regulates angiogenesis. In another nonexclusive embodiment, the MRD target is a factor that regulates cellular adhesion and/or cell-cell interaction. In certain non-exclusive embodiments, the MRD target is a cell signaling molecule. In another nonexclusive embodiment, the MRD target is a factor that regulates one or more immune responses, such as, autoimmunity, inflammation and immune responses against cancer cells. In another nonexclusive embodiment, the MRD target is a factor that regulates cellular adhesion and/or cell-cell interaction. In an additional nonexclusive embodiment, the MRD target is a cell signaling molecule. In another embodiment, an MRD can bind a target that is itself an MRD. The ability of MRDs to bind a target and block, increase, or interfere with the biological activity of the MRD target can be determined using or routinely modifying assays, bioassays, and/or animal models known in the art for evaluating such activity.

The MRDs are able to bind their respective target when the MRDs are attached to an antibody. In some embodiments, the MRD is able to bind its target when not attached to an antibody. In some embodiments, the MRD is a target agonist. In other embodiments, the MRD is a target antagonist. In certain embodiments, the MRD can be used to localize an MRD-containing antibody to an area where the MRD target is located.

The sequence of the MRD can be determined several ways. For example, MRD sequences can be derived from natural ligands or known sequences that bind to a specific target binding site. Additionally, phage display technologies have emerged as a powerful method in identifying peptides which bind to target receptors and ligands. In peptide phage display libraries, naturally occurring and non-naturally occurring (e.g., random peptide) sequences can be displayed by fusion with coat proteins of filamentous phage. The methods for elucidating binding sites on polypeptides using phage display vectors has been previously described, in particular in WO94/18221, which is herein incorporated by reference. The methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing polypeptides that bind to the pre-selected target site of interest.

The methods of the present invention for preparing MRDs include the use of phage display vectors for their particular advantage of providing a means to screen a very large population of expressed display proteins and thereby locate one or more specific clones that code for a desired target binding reactivity. The ability of the polypeptides encoded by the clones to bind a target and/or alter the biological activity of the target can be determined using or routinely modifying assays and other methodologies described herein or otherwise known in the art. For example, phage display technology can be used to identify and improve the binding properties of MRDs. See, e.g., Scott et al., Science 249:386 (1990); Devlin et al., Science 249:404 (1990); U.S. Pat. Nos. 5,223,409, 5,733,731, 5,498,530, 5,432,018, 5,338,665, 5,922,545; and Int. Appl. Publ. Nos. WO96/40987 and WO98/15833; which are herein incorporated by reference. In peptide phage display libraries, natural and/or non-naturally occurring peptide sequences can be displayed by fusion with coat proteins of filamentous phage. The displayed peptides can be affinity-eluted against a target of interest if desired. The retained phage may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al., Science 276:1696-9 (1997), in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424 (1997).

Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide such as an MRD may be designed. See, e.g., Takasaki et al., Nature Biotech. 15:1266-1270 (1997). These analytical methods may also be used to investigate the interaction between a target and an MRD selected by phage display, which can suggest further modification of the MRDs to increase binding affinity.

Other methods known in the art can be used to identify MRDs. For example, a peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). These and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. This and related methods are collectively referred to as “ribosome display.” Other known methods employ chemical linkage of peptides to RNA. See, for example, Roberts and Szostak, Proc. Natl. Acad. Sci. USA 94:12297-12303 (1997). This and related methods are collectively referred to as “RNA-peptide screening, RNA display and mRNA display.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. These and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells and Lowman, Curr. Opin. Biotechnol. 3:355-362 (1992). Furthermore, constrained libraries, linear libraries, and/or focused libraries (comprised of structurally related domains that share significant primary sequence homology) can be used to identify, characterize, and modify MRDs

An improved MRD that specifically binds a desired target can also be prepared based on a known MRD sequence. For example, at least one, two, three, four, five, or more amino acid mutations (e.g., conservative or non-conservative substitutions), deletions or insertions can be introduced into a known MRD sequence and the resulting MRD can be screened for binding to the desired target and biological activity, such as the ability to antagonize target biological activity or to agonize target biological activity. In another embodiment, the sites selected for modification are affinity matured using phage display techniques known in the art. See, e.g., Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-4 24 (1997).

Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purposes of making amino acid addition(s), substitution(s) or deletion(s) in the antibody sequence, or for creating/deleting restriction sites and sequences coding for desired amino acids (e.g., cysteine) to facilitate further manipulations. Such techniques include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985); Hutchinson et al., J. Biol. Chem. 253:6551 (1978)), oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet. 19:423-463 (1985); Hill et al., Methods Enzymol. 155:558-568 (1987)), PCR-based overlap extension (Ho et al., Gene 77:51-59 (1989)), PCR-based megaprimer mutagenesis (Sarkar et al., Biotechniques 8:404-407 (1990)), etc. Modifications can be confirmed by DNA sequencing.

Additional fusion proteins can be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling can be employed to alter the activities of SYNAGIS® or fragments thereof (e.g., an antibody or a fragment thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama et al., Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); Lorenzo et al., Biotechniques 24(2):308-313 (1998); U.S. Appl. Publ. Nos. 20030118592 and 200330133939; and Int. Appl. Publ. No. WO02/056910; each of which is herein incorporated by reference in its entirety.

Additionally, MRDs can be identified based on their effects in assays that measure particular pathways or activities. For example, assays that measure signaling pathways (e.g., phosphorylation studies or multimerization), ion channel fluxes, intracellular cAMP levels, cellular activities such as migration, adherence, proliferation, or apoptosis, and viral entry, replication, budding, or integration can be used to identify, characterize, and improve MRDs.

Variants and derivatives of the MRDs that retain the ability to bind the target antigen are included within the scope of the present invention. Included within variants are insertional, deletional, and substitutional variants, as well as variants that include MRDs presented herein with additional amino acids at the N- and/or C-terminus, including from about 0 to 50, 0 to 40, 0 to 30, 0 to 20 amino acids and the like. It is understood that a particular MRD of the present invention may be modified to contain one, two, or all three types of variants. Insertional and substitutional variants may contain natural amino acids, unconventional amino acids, or both. In some embodiments, the MRD contains a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid differences when compared to an MRD sequence described herein. In some embodiments, the amino acid differences are substitutions. These substitutions can be conservative or non-conservative in nature and can include unconventional or non-natural amino acids. In other embodiments the MRD contains a sequence that competitively inhibits the ability of an MRD-containing sequence described herein to bind with a target molecule. The ability of an MRD to competitively inhibit another MRD-containing sequence can be determined using techniques known in the art, including ELISA and BIAcore analysis.

The ability of an MRD to bind its target can be assessed using any technique that assesses molecular interaction. For example, MRD-target interaction can be assayed as described in the Examples below or alternatively, using in vitro or in vivo binding assays such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC). Assays evaluating the ability of an MRD to functionally affect its target (e.g., assays to measure signaling, proliferation, migration etc.) can also be used to indirectly assess MRD-target interaction.

An improved MRD that has a particular half-life in vivo can also be prepared based on a known MRD sequence. For example, at least one, two, three, four, five, or more amino acid mutations (e.g., conservative or non-conservative substitutions), deletions or insertions can be introduced into a known MRD sequence and the resulting MRD can be screened for increased half-life. Thus, variants and derivatives of the MRDs that retain the ability to bind the target and have an increased half-life can be included in multivalent and multispecific compositions (e.g., MRD-containing antibodies). Thus, in some embodiments, an MRD in an MRD-containing antibody has a half-life of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, or at least about 150 hours. In some embodiments, an MRD in an MRD-containing antibody has a half-life of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, or at least about 150 hours.

Once the sequence of the MRD has been elucidated, the peptides may be prepared by any of the methods known in the art. For example, the MRD peptides can be chemically synthesized and operably attached to the antibody or can be synthesized using recombinant technology. For example, MRDs can be synthesized in solution or on a solid support using known techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Tam et al., J. Am. Chem. Soc. 105:6442 (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int. J. Pep. Protein Res., 30:705-739 (1987); and U.S. Pat. No. 5,424,398, each of which is incorporated herein by reference in its entirety.

MRDs can be synthesized with covalently attached molecules that are not amino acids but aid in the purification, identification, and/or tracking of an MRD in vitro or in vivo. (e.g., biotin for reacting with avidin or avidin-labeled molecules).

The following MRD targets are described in more detail by way of example only.

In some embodiments described herein, the MRD targets an integrin. The role of integrins such as αvβ3 and αvβ5 as tumor-associated markers has been well documented. A recent study of 25 permanent human cell lines established from advanced ovarian cancer demonstrated that all lines were positive for αvβ5 expression and many were positive for αvβ3 expression. Studies have also shown that αvβ3 and αvβ5 is highly expressed on malignant human cervical tumor tissues. Integrins have also demonstrated therapeutic effects in animal models of Kaposi's sarcoma, melanoma, and breast cancer.

A number of integrin αvβ3 and αvβ5 antagonists are in clinical development. These include cyclic RGD peptides and synthetic small molecule RGD mimetics. Two antibody-based integrin antagonists are currently in clinical trials for the treatment of cancer. The first is VITAXIN® (MEDI-522, Abegrein), the humanized form of the murine anti-human αvβ3 antibody LM609. A dose-escalating phase I study in cancer patients demonstrated that VITAXIN® is safe for use in humans. Another antibody in clinical trials is CNT095, a fully human Ab that recognizes αv integrins. A Phase I study of CNT095 in patients with a variety of solid tumors has shown that it is well tolerated. Cliengitide (EMD 121974), a peptide antagonist of αvβ3 and αvβ5, has also proven safe in phase I trials. Furthermore, there have been numerous drug targeting and imaging studies based on the use of ligands for these receptors. These preclinical and clinical observations demonstrate the importance of targeting αvβ3 and αvβ5 and studies involving the use of antibodies in this strategy have consistently reported that targeting through these integrins is safe.

Clinical trials are also ongoing for antagonists targeting α5vβ1 for treating metastatic melanoma, renal cell carcinoma, and non-small cell lung cancer (M200 (volociximab) and malignant glioma (ATN-161).

Integrin-binding MRDs containing one or more RGD tripeptide sequence motifs represent an example of MRDs of the invention. Ligands having the RGD motif as a minimum recognition domain and from which MRDs of the invention can be derived are well known, a partial list of which includes, with the corresponding integrin target in parenthesis, fibronectin (α3β1, α5β1, αvβ1, α11β3, αvβ3, and α3β1) fibrinogen (αMβ2 and α11bβ1) von Willebrand factor (α11bβ3 and αvβ3), and vitronectin (α11bβ3, αvβ3 and αvβ5).

In one embodiment, the RGD containing targeting MRD is a member selected from the group consisting of: YCRGDCT (SEQ ID NO:3); PCRGDCL (SEQ ID NO:4); TCRGDCY (SEQ ID NO:5); and LCRGDCF (SEQ ID NO:6).

A MRD that mimics a non-RGD-dependent binding site on an integrin receptor and having the target binding specificity of a high affinity ligand that recognizes the selected integrin is also contemplated in the present invention. MRDs that bind to an integrin receptor and disrupt binding and/or signaling activity of the integrin are also contemplated.

In some embodiments, the MRD targets an angiogenic molecule. Angiogenesis is essential to many physiological and pathological processes. Ang2 has been shown to act as a proangiogenic molecule. Administration of Ang2-selective inhibitors is sufficient to suppress both tumor angiogenesis and corneal angiogenesis. Therefore, Ang2 inhibition alone or in combination with inhibition of other angiogenic factors, such as VEGF, can represent an effective antiangiogenic strategy for treating patients with solid tumors.

It is contemplated that MRDs useful in the present invention include those that bind to angiogenic receptors, angiogenic factors, and/or Ang2. In a specific embodiment, an MRD of the invention binds Ang2. In further embodiments, the TIE2 binding component comprises a fragment of ANG2 that binds TIE2. In particular embodiments, compositions of the invention bind TIE2 and comprise amino acids 283-449 of the human ANG2 disclosed in NCBI Ref. Seq. No. NP_001138.1.

In one embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds Ang2 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:136-619 and 620, as set forth in Table 10. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for Ang2 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 136-619 and 620. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of Ang2 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:136-619 and 620.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds Ang2 and contains an amino acid sequence selected from the group consisting of: DCFWYPNPEWCY (ANG100; SEQ ID NO:621); KCPLLPDWASCH (ANG99; SEQ ID NO:622); TCQDYPFWRYCH (ANG53; SEQ ID NO:623); DCYF YPNPPHCY (ANG57; SEQ ID NO:624); PHEECYFYPNPPHCYTMS (ANG318; SEQ ID NO:625); DCAVYPNPPWCYKMEFGK (ANG202; SEQ ID NO: 626); RPILCPLLPDW ISCHEWL (ANG205; SEQ ID NO:627); and LWDDCYFFPNPPHCYNSP (ANG200; SEQ ID NO:628). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for Ang2 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 621-627 and 628. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of Ang2 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:621-627 and 628.

In one embodiment, an MRD and/or MRD-containing antibody binds Ang2 and contains a sequence selected from the group consisting of: GAQTNFMPMDDLEQRLYEQFI LQQGLE (SEQ ID NO:9) (ANGa); LWDDCYFFP NPPHCYNSP (SEQ ID NO:11) (ANGb); LWDDCYSYPNPPHCYNSP (SEQ ID NO:12) (ANGc); LWDDCYSFPNPPHCYNSP (SEQ ID NO:15) (ANGd); DCAVYPNPPWCYKME FGK (SEQ ID NO:16) (ANGe); PHEECYFY PNPPHCYTMS (SEQ ID NO:17) (ANGf); and PHEECYSYPNPPHCYTMS (SEQ ID NO:18) (ANGg).

In another embodiment, an MRD-containing antibody comprises an ANG-2 binding peptide disclosed in U.S. Pat. Nos. 7,309,483, 7,205,275, 7,138,370 7,063,965, 7,063,840, 7,045,302, 7,008,781, 6,825,008, 6,645,484, 6,627,415, 6,455,035, 6,441,137, 6,433,143, 6,265,564, 6,166,185, 5,879,672, 5,814,464, 5,681,714, 5,650,490, 5,643,755 and 5,521,073; and U.S. Appl. Publ. Nos. 2007/0225221, 2007/0093419, 2007/0093418, 2007/0072801, 2007/0025993, 2006/0122370, 2005/0186665, 2005/0175617, 2005/0106099, 2005/0100906, 2003/0236193, 2003/0229023, 2003/0166858, 2003/0166857, 2003/0162712, 2003/0109677, 2003/0092891, 2003/0040463, 2002/0173627 and 2002/0039992, and Intl. Appl. Publ. Nos. WO2006/005361, WO/2006/002854, WO2004/092215, WO/2004/076650, WO2003/057134, WO/2000/075323, WO2000/065085, WO/1998/018914 or WO1995/021866, the disclosures of each of which is herein incorporated by reference in its entirety.

In one embodiment, an MRD-containing antibody contains an MRD that preferentially binds ANG2 over ANG1 and ANG4. In a further embodiment, the MRD-containing antibody contains an MRD having a sequence selected from the group consisting of: ANG100 (SEQ ID NO:621), ANG156 (SEQ ID NO:255), ANG318 (SEQ ID NO:625) and ANG599 (SEQ ID NO:367). In an additional embodiment, an MRD-containing antibody contains an MRD that preferentially binds ANG1 and ANG2 over ANG4. In a further embodiment, the MRD-containing antibody contains an MRD having a sequence selected from the group consisting of: ANG126 (SEQ ID NO:232), ANG129 (SEQ ID NO:234), ANG179 (SEQ ID NO:270), ANG200 (SEQ ID NO:628), ANG303 (SEQ ID NO:331) and ANG335 (SEQ ID NO:332). In another embodiment, an MRD-containing antibody contains an MRD that binds ANG1, ANG2 and ANG4. In a further embodiment, the MRD-containing antibody contains an MRD having the sequence of ANG335 (SEQ ID NO:332). MRD-containing antibodies that compete for ANG1, ANG2 and/or ANG4 binding with one or more of the above MRDs and/or MRD-containing antibodies are also encompassed by the invention.

In some embodiments, the MRD targets vascular endothelial growth factor (VEGF). In one embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds VEGF and contains an amino acid sequence selected from the group consisting of: SEQ ID NOs:629-691 and 692, as set forth in Table 11. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for VEGF binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:629-691 and 692. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of VEGF as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:629-691 and 692.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds VEGF and contains an amino acid sequence selected from the group consisting of: ACVKQVPWWLCI (VGF5; SEQ ID NO:693); TCWKPTPWWLCD (VGF26; SEQ ID NO:694); WCNGFPPNYPCY (VGF57; SEQ ID NO:695); GCVKEAPWWLCV (VGF61; SEQ ID NO:696); and WCNGFPANYPCY (VGF50; SEQ ID NO:697). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for VEGF binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:693-696 and 697. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of VEGF as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:693-696 and 697.

In one embodiment, the antibody-MRD fusion comprises an MRD with the sequence ATWLPPP (SEQ ID NO:71), which inhibits VEGF-mediated angiogenesis. Binetruy-Tournaire et al., EMBO J. 19:1525-1533 (2000). In additional embodiments, an anti-VEGF antibody containing an MRD that targets VEGF is contemplated in the present invention. Anti-VEGF antibodies can be found for example in Presta et al., Cancer Research 57:4593-4599 (1997); and Fuh et al., J. Biol. Chem. 281:10 6625 (2006), each of which is herein incorporated by reference in its entirety.

Insulin-like growth factor-I receptor (IGF1R)-specific MRDs can also be used in the present invention. In one embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds IGF1R and contains an amino acid sequence selected from the group consisting of: SEQ ID NOs:698-1149 and 1150, as set forth in Table 12. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IGF1R binding with a polypeptide having an amino acid sequence selected from the group consisting of: NO:698-1149 and 1150. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IGF1R as a polypeptide having an amino acid sequence selected from the group consisting of: NO:698-1149 and 1150.

In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IGF1R binding with a polypeptide having an amino acid sequence selected from the group consisting of: LCKEFPELCF (IGF252; SEQ ID NO:1151); QPWEGYSWLY (IGF71; SEQ ID NO:1152); RITPSDLCKEFPELCF (IGF550; SEQ ID NO:1153); GFHHAYYKY RGQAGQGSATGGSSRITPSDLCKEFP ELCF (IGF2044; SEQ ID NO:1154); LCHGYPWYGPCQGQAGQGSATGGSGSTAS SRITPSDLCKEFPELCF (IGF2037: SEQ ID NO:1155); GSWCDHYPQPVMCLGQAG QGSATGGSSRITPSDLCKEFPELCF (IGF2039 SEQ ID NO:1156); and TFENAL YCLAYGICDKITLI (IGF2045; SEQ ID NO:1157). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IGF1R binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1151-1156 and 1157. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IGF1R as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: SEQ ID NO:1151-1156 and 1157.

Vascular homing-specific MRDs are also contemplated for use in the present invention. A number of studies have characterized the efficacy of linking the vascular homing peptide to other proteins like IL12 or drugs to direct their delivery in live animals.

Numerous other target binding sites are contemplated as being the target of the antibody-MRD fusions of the present invention, including for example, FGFR1, FGFR2, EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-1 receptor, and hepatocyte growth factor receptor. MRDs can be directed towards these target binding sites or the corresponding ligands.

In one embodiment, the MRD binds to IL6. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds IL6 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:1158-1731 and 1732, as set forth in Table 13. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL6 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1158-1731 and 1732. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL6 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1158-1731 and 1732.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds IL6 and contains an amino acid sequence selected from the group consisting of: YCPWDPIMMQCA (ILB394; SEQ ID NO:1733); LSNSCAW DPLLMNCIDTH (ILB227; SEQ ID NO:1734); FNEDCMWDPLLMDCAYSP (ILB915; SEQ ID NO:1735); INNLSPVCYWDPLLMDCI (ILB235; SEQ ID NO:1736); VQY WEFETWEGQAGQGSFNEDCMWDPLLMDCAYSP (ILB914; SEQ ID NO:1737); HQADDPFGQWGQAGQGSFNEDCMWDPLLMDCAYSP (ILB910; SEQ ID NO:1738); PLWEQTKWQYGQAGQGSFNEDCMWDPLLMDCAYSP (ILB916; SEQ ID NO:1739); FNEDC MWDPLLMDCAYSPGQGSATGGSAAGGGSMCLTYEEICS (ILB790; SEQ ID NO:1740); ISMPCH SWEHCLSLL (ILB754; SEQ ID NO:1741); GWGRVCDADYCCWVVC (ILB753; SEQ ID NO:1742); EYDWCMWEVK MFEEACWSLS (ILB909; SEQ ID NO:1743); VLSECFEAYRVVCQALT (ILB913; SEQ ID NO:1744); MGIRCQTWDHCLSIL (ILB911; SEQ ID NO:1745); and QRYPCTTWEDCLVVL (ILB912; SEQ ID NO:1746). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL6 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1733-1745 and 1746 In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL6 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1733-1745 and 1746.

In one embodiment, the MRD binds to IL6R. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds IL6R and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:1747-1908 and 1909, as set forth in Table 14. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL6R binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1747-1908 and 1909. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL6R as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1747-1908 and 1909.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds IL6R and contains an amino acid sequence selected from the group consisting of: DLLYCEWEMVVRECHGTI (ILC111; SEQ ID NO:1910); EFVLCELAM VFRECHGAV (ILC82; SEQ ID NO:1911); ELIFCDLEMVFNECHGAI (ILC85; SEQ ID NO:1912); DYILCDLEMVFRECHGVL (ILC289; SEQ ID NO:1913); EFILCELEMVIRECHGTT (ILC92; SEQ ID NO:1914); PCE EAFAAYLE (ILC110; SEQ ID NO:1915); PCEEAFAAYLEEAYN (ILC134; SEQ ID NO:1916); ACEEAFASYLEENVM (ILC100; SEQ ID NO:1917); DCNALFTGDLAW (ILC145; SEQ ID NO:1918); DEEWTCFP TLCSLWSE (ILC51; SEQ ID NO:1919); EEEWTCFPSLCTIWSQ (ILC163; SEQ ID NO:1920); EEEWTCFPYLCSDWLQ (ILC168; SEQ ID NO:1921); EEEWTCFPYLCSEWVH (ILC235; SEQ ID NO:1922); QEEWTCFPYLCSYWAQ (ILC217; SEQ ID NO:1923); GQDDCWPYYCDELEY (ILC54 SEQ ID NO:1924); SCWTCIPEMVNCEAAH (ILC293; SEQ ID NO:1925); YWAKCDYHEGWHHCELHP (ILC296; SEQ ID NO:1926); VFWDCWYYGT WIECENTG (ILC295; SEQ ID NO:1927); GDTPCQEWPYWCLPPY (ILC290; SEQ ID NO:1928); HLISCEFHEKYVECVEVA (ILC291; SEQ ID NO:1929); STDYC EVLEIQWVCYRPP (ILC294; SEQ ID NO:1930); and QIVECWTEMDWHHCVLFF (ILC292 SEQ ID NO:1931). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL6R binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1910-1930 and 1931. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL6R as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1910-1930 and 1931.

In one embodiment, the MRD binds to IL17a. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds IL17a and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:1932-1989 and 1990, as set forth in Table 15. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL17a binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1932-1989 and 1990. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL17a as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1932-1989 and 1990.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds IL17a and contains the amino acid sequence LSLWCWLINDVNCSEPV (ILA72; SEQ ID NO:1991) or NPLWCWMFPADDPC VHPG (ILA20; SEQ ID NO:1992). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for IL17a binding with a polypeptide having the amino acid sequence of SEQ ID NO:1991 or SEQ ID NO:1992. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of IL17a as a polypeptide having the amino acid sequence of SEQ ID NO:1991 or SEQ ID NO:1992.

In one embodiment, the MRD binds to TNFSF15 (TL1A). In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds TL1A and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:1993-2234 and 2235, as set forth in Table 16. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for TL1A binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1993-2234 and 2235. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of TL1A as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:1993-2234 and 2235.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds TL1A and contains an amino acid sequence selected from the group consisting of: QDRVCWDFHQKLQFCELSM (TLA525; SEQ ID NO:2236); YSSVCWDDTRE ALFYCETTV (TLA27; SEQ ID NO:2237); EYALCWVEAQEK LEWCKGLN (TLA522; SEQ ID NO:2238); DYEVCWDVHVDRLVYCDAPM (TLA523; SEQ ID NO:2239); STEVCWDFIEQKLIWCSTFR (TLA35; SEQ ID NO:2240); QSDVCW NQSHQLLQFCWESN (TLA54; SEQ ID NO:2241); and DYAVCWDPDYG NLVWCNVLS (TLA524; SEQ ID NO:2242). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for TL1A binding with a polypeptide having an amino acid sequence selected from the group consisting of: 2236-2241 and 2242. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of TL1A as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:2236-2241 and 2242.

In one embodiment, the MRD binds to TNFSF13B (BLyS). In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds BLyS and contains an amino acid sequence selected from the group consisting of: GDMGCWDWLTRTQVKCSLLV (BLY50; SEQ ID NO:2243); PRGTCWADPLT GQCVQVM (BLY47; SEQ ID NO:2244); PAGMCFDNLTLQMVQCTSLK (BLY35 SEQ ID NO:2245); GPWACYETPTRQMCVPVF (BLY69 SEQ ID NO:2246); ERKA CYFDPLVK QCLFVR (BLY60; SEQ ID NO:2247); TMPTCFMDPLTHQCWPSV (BLY34; SEQ ID NO:2248); HPGRCYDQLTYEWVTCWHLW (BLY31; SEQ ID NO:2249); RADLCYHEHH NNECFFGM (BLY73; SEQ ID NO:2250); and TGSSC WDILTKQMVPC LTAW (BLY27; SEQ ID NO:2251). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for BLyS binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2243-2250 and 2251. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of BLyS as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:2243-2250 and 2251.

In one embodiment, the MRD binds to HER2/3.

In another embodiment, the MRD binds EGFR. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds EGFR and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:2252-3090 and 3091, as set forth in Table 17. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for EGFR binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:2252-3090 and 3091. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of EGFR as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:2252-3090 and 3091.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds EGFR and contains an amino acid sequence selected from the group consisting of: LTAPCYWTTWGKECLMLR (EGA79; SEQ ID NO:3092); LNDNCYWTTWGRECYLLR (EGA191; SEQ ID NO:3093); FSDPCYWTTWGR ECFLLP (EGA214; SEQ ID NO:3094); LPEGCYWTTWGKECLLLP (EGA234; SEQ ID NO:3095); LPQGCVWTNWGKECYLPL (EGA626; SEQ ID NO:3096); LPEGCW WSQWGKECLLLP (EGA623; SEQ ID NO:3097); LPQECYWAHWGKECYLPP (EGA625; SEQ ID NO:3098); LPEGCWWAPWGKECLLHP (EGA673 SEQ ID NO:3099); LPEDCWWSTWGRECLLPP (EGA990; SEQ ID NO:3100); ADSWCHTL YWQLHHCASWE (EGA349; SEQ ID NO:3101); EESLCHTLYRQLHNCRSLE (EGA406; SEQ ID NO:3102); ADSLCLTEYLELLQCERWE (EGA466; SEQ ID NO:3103); ADSLCQTRHQQLLDCERQE (EGA467; SEQ ID NO:3104); PESWCHTLY WNLQHCLSQE (EGA433; SEQ ID NO:3105); YAEHCWQFPTDWICTLMT (EGA518; SEQ ID NO:3106); YAEHCLMFPGDWICTLLP (EGA771; SEQ ID NO:3107); YASHCSQFPGDWICSLMT (EGA772; SEQ ID NO:3108); YAEHCRQ FPSDWICTLLP (EGA797; SEQ ID NO:3109); YADHCSQFPNDWICTLLS (EGA926; SEQ ID NO:3110); YAEHCSLFPTDWICTLMS (EGA958; SEQ ID NO:3111); and YAEHCSQFPSDWICSLLS (EGA917; SEQ ID NO:3112). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for EGFR binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:3092-3111 and 3112. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of EGFR as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:3092-3111 and 3112.

In another embodiment, the MRD binds ErbB2. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds ErbB2 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 3113-3494 and 3495 as set forth in Table 18. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for ErbB2 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 3113-3494 and 3495. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of ErbB2 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 3113-3494 and 3495.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds ErbB2 and contains an amino acid sequence selected from the group consisting of: SAISCYVYQQHRHCDWDH (EGB417; SEQ ID NO:3496); WSGYCEMKDHWAHCGHSE (EGB390; SEQ ID NO:3497); and WSGYCETPSGWK ACRGNI (EGB858; SEQ ID NO:3498). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for ErbB2 binding with a polypeptide having the amino acid sequence of SEQ ID NO:3496, SEQ ID NO:3497 or SEQ ID NO:3498. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of ErbB2 as a polypeptide having the amino acid sequence of SEQ ID NO:3496, SEQ ID NO:3497 or SEQ ID NO:3498.

In another embodiment, the MRD binds ErbB3. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds ErbB3 contains an amino acid sequence selected from the group consisting of: SEQ ID NO:3499-4086 and 4087, as set forth in Table 19. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for ErbB3 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:3499-4086 and 4087. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of ErbB3 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:3499-4086 and 4087.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds ErbB3 and contains an amino acid sequence selected from the group consisting of: SSETYCPFLNPYWYDQCMSRT (EGC1077; SEQ ID NO:4088); SEPYCPFLNPTWKSQCIART (EGC180; SEQ ID NO:4089); SQVSCP FQNPIWWSHCMELM (EGC42; SEQ ID NO:4090); GSKPYCPFTNPIWYDECIKRS (EGC593; SEQ ID NO:4091); ATEPYCPFLNPIWKAECLARS (EGC503; SEQ ID NO:4092); KFLMCQVYPSEKWHVCVETL (EGC41 SEQ ID NO:4093); and SKPYC PFTNPIWYDECIKRSGGGSGGGSQAAAGGSYMHEPHMQVLEIMN (EGC1086; SEQ ID NO:4094). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for ErbB3 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4088-4093 and 4094. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of ErbB3 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4088-4093 and 4094.

Insulin-like growth factor-I receptor (ErbB4)-specific MRDs can also be used in the present invention. In one embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds ErbB4 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:4095-4123 and 4124, as set forth in Table 20. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for ErbB4 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4095-4123 and 4124. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of ErbB4 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4095-4123 and 4124.

In another embodiment, the MRD binds TNFRSF10B (DR5). In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds DR5 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:4125-4319 and 4320, as set forth in Table 21. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for DR5 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4125-4319 and 4320. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of DR5 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4125-4319 and 4320.

In a particular embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds DR5 and contains the amino acid sequence of WDCLGKLGQR QCVKL (DR5218; SEQ ID NO:4321) or FPRPCAEEDVIMTCRWRV (DR53; SEQ ID NO:4322). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for DR5 binding with a polypeptide having the amino acid sequence of SEQ ID NO:4321 or SEQ ID NO:4322. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of DR5 as a polypeptide having the amino acid sequence of SEQ ID NO:4321 or SEQ ID NO:4322. As demonstrated herein, increasing the valency of DR5 MRDs in an MRD-containing antibody results in a corresponding increase in DR-5 mediated cell cytotoxicity (see, Example 46). Thus, in additional embodiments, the invention encompasses an MRD-containing antibody that contains at least 4, at least 6, at least 8 or at least 10 MRDs that bind DR5. Additionally, as demonstrated herein, increasing the multi-epitopic targeting for a target other than DR5 (e.g., ErbB2 and EGFR) in a DR5-binding MRD-containing antibody correlates with increased DR5 mediated cell cytotoxicity Thus, in additional embodiments, the invention encompasses an MRD-containing antibody that binds DR5 and at least two distinct epitopes of a target other than DR5. Moreover, as demonstrated herein, the antibody scaffold of a DR5-binding MRD containing antibody influences the level of DR5 mediated cell cytotoxicity of the MRD-containing antibody. In some embodiments, the antibody combining site of the DR5-binding MRD-containing antibody binds a molecule on the surface of a targeted cell expressing DR5. In a further embodiment, the antibody combining site of the MRD-containing antibody binds a molecule on the surface of a cell and MRDs of the MRD-containing antibody binds DR5 through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In alternative embodiments, the antibody combining site of a DR5-binding MRD-containing antibody does not bind to a molecule on the surface of a targeted cell that expresses DR5 on its surface. In a further embodiment, the antibody combining site of the MRD-containing antibody does not bind a molecule on the surface of a targeted cell and MRDs of the MRD-containing antibody binds DR5 through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

In additional embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds DR4 (TNFRSF10A/TRAIL R1). In further embodiments, the MRD-containing antibody contains at least 4, at least 6, at least 8 or at least 10 MRDs that bind DR4 (TNFRSF10A/(TRAIL R1). In additional embodiments, the invention encompasses an MRD-containing antibody that binds DR4 and at least two distinct epitopes of a target other than DR4. In some embodiments, the antibody combining site of the DR4-binding MRD-containing antibody binds a molecule on the surface of a targeted cell expressing DR4. In a further embodiment, the antibody of the MRD-containing antibody binds a molecule on the surface of a cell and MRDs of the MRD-containing antibody binds DR4 through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In alternative embodiments, the antibody combining site of a DR4-binding MRD-containing antibody does not bind to a molecule on the surface of a targeted cell that expresses DR4 on its surface. In a further embodiment, the antibody combining site of the MRD-containing antibody does not bind a molecule on the surface of a targeted cell and MRDs of the MRD-containing antibody binds DR4 through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

In further embodiments, the invention encompasses an MRD-containing antibody that binds a cell surface signaling molecule. In further embodiments, the MRD-containing antibody contains at least 4, at least 6, at least 8 or at least 10 MRDs that bind the cell surface signaling molecule. In particular embodiments, the cell surface signaling molecule is a member selected from: c-Met, EGFR, ErbB2, VEGFR1, VEGFR2, VEGFR3, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, PDGFRA, PDGFRB, netrin, CD28, TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF6 (Fas, CD95), TNFRSF21 or TNFRSF25, TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF11A (RANK), TNFRSF21 (DR6), TNFRSF25 (DR3) and LRP6. In additional embodiments, the invention encompasses an MRD-containing antibody that binds the cell surface signaling molecule and at least two distinct epitopes of a target other than the cell surface signaling molecule. In some embodiments, the antibody combining site of the MRD-containing antibody binds a molecule on the surface of a targeted cell expressing the cell surface signaling molecule. In a further embodiment, the antibody of the MRD-containing antibody binds a molecule on the surface of a cell and MRDs of the MRD-containing antibody binds the cell surface signaling molecule through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In alternative embodiments, the antibody combining site of a cell surface signaling molecule-binding MRD-containing antibody does not bind to a molecule on the surface of a targeted cell that expresses the cell surface signaling molecule. In a further embodiment, the antibody combining site of the MRD-containing antibody does not bind a molecule on the surface of a targeted cell and MRDs of the MRD-containing antibody binds the cell surface signaling molecule through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

In another embodiment, the MRD binds CD19. In some embodiments, the invention encompasses an MRD and/or MRD-containing antibody that binds CD19 and contains an amino acid sequence selected from the group consisting of: STLTCQVWLEDWYECIEVD (CDA1; SEQ ID NO:4323); PNWVCWET PWTYECLEIE (CDA16; SEQ ID NO:4324); VLLH CFGEPQWMECVPYV (CDA26; SEQ ID NO:4325); FSPFCQYFQEFGECHYLS (CDA33; SEQ ID NO:4326); LLLRCM YEPYYWELQCVEVE (CDA48; SEQ ID NO:4327); and YIYTCSFIWDYQEIYCSPEL (CDA53; SEQ ID NO:4328). In an additional embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for CD19 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4323-4327 and 4328. In a further embodiment, the invention encompasses an MRD and/or MRD-containing antibody that binds to the same epitope of CD19 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4323-4327 and 4328.

In some embodiments, the MRD binds to a human protein. In some embodiments, the MRD binds to both a human protein and its ortholog in mouse, rat, rabbit, or hamster.

III. Antibodies

The antibody in the multivalent and multispecific compositions (e.g., MRD-containing antibodies) described herein can be any suitable antigen-binding immunoglobulin. In certain embodiments, the MRD-containing antibody molecules described herein retain the structural and functional properties of traditional monoclonal antibodies. Thus, the antibodies retain their epitope binding properties, but advantageously also incorporate one or more additional target-binding specificities.

Antibodies that can be used in the multivalent and multispecific compositions (e.g., MRD-containing antibodies) include, but are not limited to, monoclonal, multispecific, human, humanized, primatized, and chimeric antibodies. Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In specific embodiments, the antibodies are IgG1. In other specific embodiments, the antibodies are IgG3.

Antibodies that can be used as part of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be naturally derived or the result of recombinant engineering (e.g., phage display, xenomouse, and synthetic). The antibodies can include modifications, for example, to enhance half-life or to increase or decrease antibody dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity. Antibodies can be from or derived from any animal origin including birds and mammals or generated synthetically. In some embodiments, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In specific embodiments, the antibodies are human.

In certain embodiments, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. In alternative embodiments, the heavy chain portion-containing monomers of the invention are not identical. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.

Bispecific, bivalent antibodies, and methods of making them, are described, for instance in U.S. Pat. Nos. 5,731,168, 5,807,706, 5,821,333, and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537; each of which is herein incorporated by reference in its entirety. Bispecific tetravalent antibodies, and methods of making them are described, for instance, in Int. Appl. Publ. Nos. WO02/096948 and WO00/44788, the disclosures of both of which are herein incorporated by reference in its entirety. See generally, Int. Appl. Publ. Nos. WO93/17715, WO92/08802, WO91/00360, and WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992).

The heavy chain portions of the antibody component of the MRD-antibody fusions for use in the methods disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

In some embodiments, the antigen binding domains of the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind to their target with a dissociation constant or Kd of less than 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰, M, 10⁻¹⁰ M, 4×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. In one embodiment, the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd of less than 5×10⁻⁵ M. In another embodiment, antigen binding of the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) has a dissociation constant or Kd of less than 5×10⁻⁸ M. In another embodiment, antigen binding of the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) has a dissociation constant or Kd of less than less than 5×10⁻⁹ M. In another embodiment, the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd of less than 5×10⁻¹⁰ M. In another embodiment, the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd of less than 5×10⁻¹¹ M. In another embodiment, the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have a dissociation constant or Kd of less than 5×10⁻¹² M.

In specific embodiments, the antibody component of the MRD-containing antibody binds its target with an off rate (k_(off)) of less than 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³ sec⁻¹. More preferably, the antibody component of the MRD-containing antibody binds its target with an off rate (k_(off)) of less than 5×10⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹, or 10⁻⁷ sec⁻¹.

In other specific embodiments, the antibody component of the MRD-containing antibody binds its target with an on rate (k_(on)) of greater than 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹, or 5×10⁴ M⁻¹ sec⁻¹. More preferably, the antibody component of the MRD-containing antibody binds its target with an on rate (k_(on)) of greater than 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹, or 10⁷ M⁻¹ sec⁻¹.

Affinity maturation strategies and chain shuffling strategies (e.g., gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) are known in the art and can be employed to generate high affinity and/or to alter the activities (e.g., ADCC and CDC) of multivalent and multispecific compositions (e.g., multivalent and multispecific compositions (e.g., MRD-containing antibodies)). See, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458; and Patten et al., Curr. Opinion Biotechnol. 8:724-733 (1997), Harayama, Trends Biotechnol. 16(2):76-82 (1998), Hansson et al., J. Mol. Biol. 287:265-276 (1999) and Lorenzo and Blasco, Biotechniques 24(2):308-313 (1998); each of which is herein incorporated by reference in its entirety. Advantageously, affinity maturation strategies and chain shuffling strategies can routinely be applied to generate multivalent and multispecific compositions (e.g., MRD-containing antibodies) can also include variants and derivatives that improve antibody function and/or desirable pharmacodynamic properties.

Accordingly, certain embodiments of the invention include an antibody-MRD fusion, in which at least a fraction of one or more of the constant region domains has been altered so as to provide desired biochemical characteristics such as reduced or increased effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with an unaltered antibody of approximately the same immunoreactivity. The alterations of the constant region domains can be amino acid substitutions, insertions, or deletions.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) expressed on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to localize to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of target cells “arm” the cytotoxic cells and are required for such killing. Lysis of the target cell is extracellular, requires contact or close proximity between the cytotoxic cells and target cells, and does not involve complement.

As used herein, the term “enhances ADCC” (e.g., referring to cells) is intended to include any measurable increase in cell lysis when contacted with a variant MRD-containing antibody as compared to the cell killing of the same cell in contact with a MRD-containing antibody that has not been so modified in a way that alters ADCC in the presence of effector cells (for example, at a ratio of target cells:effector cells of 1:50), e.g., an increase in cell lysis by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or 325%.

In certain embodiments, the antibody component of the antibody-MRD fusion has been modified to increase antibody dependent cellular cytotoxicity (ADCC) (see, e.g., Bruhns et al., Blood 113:3716-3725 (2009); Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010 (2006); Stavenhagen et al., Cancer Res., 67:8882-8890 (2007); Horton et al., Cancer Res. 68:8049-8057 (2008); Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer et al., Neoplasia 11:509-517 (2009); Allan et al., WO2006/020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Watkins et al., WO2004/074455, each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that increases ADCC include one or more modifications corresponding to: IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.

In one embodiment, an Fc variant protein has enhanced ADCC activity relative to a comparable molecule. In a specific embodiment, an Fc variant protein has ADCC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold greater than that of a comparable molecule. In another specific embodiment, an Fc variant protein has enhanced binding to the Fc receptor Fc gamma RIIIA and has enhanced ADCC activity relative to a comparable molecule. In other embodiments, the Fc variant protein has both enhanced ADCC activity and enhanced serum half-life relative to a comparable molecule.

The ability of any particular Fc variant protein to mediate lysis of the target cell by ADCC can be assayed using techniques known in the art. For example, to assess ADCC activity a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) can be added to target cells in combination with immune effector cells, which can be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., J Immunol Methods 79:277-282 (1985); Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987); Wilkinson et al., J. Immunol. Methods 258:183-191 (2001); Patel et al., J. Immunol. Methods 184:29-38 (1995). Alternatively, or additionally, ADCC activity of the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998), and U.S. Pat. No. 7,662,925.

In certain embodiments, the antibody component of the antibody-MRD fusion has been modified to decrease ADCC (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., Proc. Natl. Acad. Sci. USA 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol. 200:16-26 (2000); Cole et al., Transplantation 68:563-571 (1999); Hutchins et al., Proc. Natl. Acad. Sci. USA 92:11980-11984 (1995); Reddy et al., J. Immunol. 164:1925-1933 (2000); Int. Appl. Publ. No. WO1997/11971, and WO2007/106585; U.S. Appl. Publ. 2007/0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007), each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that decreases ADCC include one or more modifications corresponding to: IgG1-K326W, E333S; IgG2-E333S; IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S, E233P, L234V, L235A; and IgG1-L234F, L235E, P331S.

In certain embodiments, the antibody component of the antibody-MRD fusion has been modified to increase antibody-dependent cell phagocytosis (ADCP); (see, e.g., Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010 (2006); Stavenhagen et al., Cancer Res., 67:8882-8890 (2007); Richards et al., Mol. Cancer Ther. 7:2517-2527 (2008); Horton et al., Cancer Res. 68:8049-8057 (2008), Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer et al., Neoplasia 11:509-517 (2009); Allan et al., WO2006/020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Watkins et al., WO2004/074455, each of which is herein incorporated by reference in its entirety.). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that increases ADCP include one or more modifications corresponding to: IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; IgG1-G236A, S239D, I332E.

In certain embodiments, the antibody component of the antibody-MRD fusion has been modified to decrease ADCP (see, e.g., Sazinsky et al., Proc. Natl. Acad. Sci. USA 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol. 200:16-20 (2000); Cole et al., Transplantation 68:563-571 (1999); Hutchins et al., Proc. Natl. Acad. Sci. USA 92:11980-11984 (1995); Reddy et al., J. Immunol. 164:1925-1933 (2000); Intl. Appl. Publ. Nos. WO1997/11971 and WO2007/106585; U.S. Appl. Publ. 2007/0148167; McEarchern et al., Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007), each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that decreases ADCC include one or more modifications corresponding to: IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S, E233P, L234V, L235A; and IgG1-L234F, L235E, P331S.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of a target cell in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule, an antibody for example, complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. In one embodiment, an Fc variant protein has enhanced CDC activity relative to a comparable molecule. In a specific embodiment, an Fc variant protein has CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, or at least 100 fold greater than that of a comparable molecule. In other embodiments, the Fc variant protein has both enhanced CDC activity and enhanced serum half-life relative to a comparable molecule.

In certain embodiments, the antibody component of the antibody-MRD fusions have been modified to increase complement-dependent cytotoxicity (CDC) (see, e.g., (see, e.g., Idusogic et al., J. Immunol. 166:2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Natsume et al., Cancer Res. 68:3863-3872 (2008), each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that increases CDC include one or more modifications corresponding to: IgG1-K326A, E333A; and IgG1-K326W, E333S, IgG2-E333S.

In one embodiment, the present invention provides formulations, wherein the Fc region comprises a non-naturally occurring amino acid residue at one or more positions selected from the group consisting of 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 as numbered by the EU index as set forth in Kabat. Optionally, the Fc region can comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, and 6,737,056; and Int. Appl. Publ. Nos. WO01/58957, WO02/06919, WO04/016750, WO04/029207, WO04/035752 and WO05/040217).

In specific embodiments MRD-containing antibodies of the invention contain an Fc variant comprising at least one non naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 200H, 299F, 299E, 313F, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A as numbered by the EU index as set forth in Kabat. Optionally, the Fc region can comprise additional and/or alternative non-naturally occurring amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, and 6,737,056; and Int. Appl. Publ. Nos. WO01/58957, WO02/06919, WO04/016750, WO04/029207, WO04/035752 and WO05/040217).

In certain embodiments, the multivalent and monovalent multispecific composition is an antibody-MRD fusions wherein the antibody component has been modified to increase inhibitory binding to Fc gamma RIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45:3926-3933 (2008)). An example of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that increases binding to inhibitory Fc gamma RIIb receptor is IgG1-S267E, L328F.

In certain embodiments, the antibody component of the antibody-MRD fusions have been modified to decrease CDC (see, e.g., Int. Appl. Publ. Nos. WO1997/11971 and WO2007/106585; U.S. Appl. Publ. No 2007/0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer 15:2739-2746 (2009); Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010 (2006); Bruckheimer et al., Neoplasia 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., Proc. Natl. Acad. Sci. USA 105:20167-20172 (2008); each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that decreases CDC include one or more modifications corresponding to: IgG1-S239D, A330L, I332E; IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234F, L235E, P331S; and IgG1-C226S, P230S.

The half-life on an IgG is mediated by its pH-dependent binding to the neonatal receptor FcRn. In certain embodiments the antibody component of the antibody-MRD fusion has been modified to enhance binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006); Dall'Acqua et al., J. Immunol. 169:5171-5180 (2002); Oganesyan et al., Mol. Immunol. 46:1750-1755 (2009); Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006), Hinton et al., J. Immunol. 176:346-356 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); Int. Appl. Publ. No. WO2006/130834; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Yeung et al., J. Immunol. 182:7663-7671 (2009); each of which is herein incorporated by reference in its entirety).

In additional embodiments, the antibody of the antibody-MRD fusion has been modified to selectively bind FcRn at pH6.0, but not pH 7.4. Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that increases half-life include one or more modifications corresponding to: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A.

In other embodiments the antibody component of the antibody-MRD fusion has been modified to decrease binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Vaccaro et al., Nat. Biotechnol. 23:1283-1288 (2005), each of which is herein incorporated by reference in its entirety). Examples of Fc sequence engineering modifications contained in the antibody component of the antibody-MRD fusions that decrease half-life include one or more modifications corresponding to: IgG1-M252Y, S254T, T256E; H433K, N434F, 436H; IgG1-1253A; and IgG1-P2571, N434H or D376V, N434H.

In some embodiments, the antibody-MRD fusions have been glyocoengineered or the Fc portion of the MRD-containing antibody has been mutated to increase effector function using techniques known in the art. For example, the inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, can easily be measured and quantified using well know immunological techniques without undue experimentation.

Methods for generating antibodies containing non-naturally occurring Fc regions are known in the art. For example, amino acid substitutions and/or deletions can be generated by mutagenesis methods, including, but not limited to, site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methods and Applications”, Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Site-directed mutagenesis can be performed by the overlap-extension PCR method (Higuchi, in “PCR Technology: Principles and Applications for DNA Amplification”, Stockton Press, New York, pp. 61-70 (1989)). Alternatively, the technique of overlap-extension PCR (Higuchi, ibid.) can be used to introduce any desired mutation(s) into a target sequence (the starting DNA). Other methods useful for the generation of antibodies containing non-naturally occurring Fc regions are known in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 5,885,573, 5,677,425, 6,165,745, 6,277,375, 5,869,046, 6,121,022, 5,624,821, 5,648,260, 6,528,624, 6,194,551, 6,737,056, 6,821,505 and 6,277,375; U.S. Appl. Publ. No. 2004/0002587 and Int Appl. Publ. Nos. WO94/29351, WO99/58572, WO00/42072, WO02/060919, WO04/029207, WO04/099249 and WO04/063351).

Multivalent and multispecific compositions (e.g., MRD-containing antibodies) used according to the methods of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, or derivatization by known protecting/blocking groups. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.

According to some embodiments the antibody component of compositions of the invention is engineered to contain one or more free cysteine amino acids having a thiol reactivity within a desirable range (e.g., 0.6 to 1.0), wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by cysteine. In some embodiments one or more free cysteine amino acid residues are located in a light chain. In additional embodiments one or more free cysteine amino acid residues are located in a heavy chain. In additional embodiments one or more free cysteine amino acid residues are located in a both the heavy and light chain. In some embodiments, the cysteine engineered MRD-containing antibody contains a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0, and a sequence modification in the light chain or the heavy chain that is disclosed in U.S. Pat. No. 7,855,275. In other embodiments, the cysteine engineered antibody contains a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0, and a sequence modification in the light chain or the heavy chain that is not disclosed in U.S. Pat. No. 7,855,275, the contents of which are herein incorporated by reference in its entirety.

In additional embodiments, the MRD-containing antibody is engineered to contain one or more free selenocysteine amino acids or another non-natural amino acid capable of forming disulfide bonds. Antibodies containing the same and methods for making such antibodies are known in the art. See, e.g., Hofer et al., Proc. Natl. Acad. Sci. 105(34):12451-12456 (2008); and Hofer et al., Biochem. 48(50):12047-12057 (2009), each of which is herein incorporated by reference in its entirety. In some embodiments one or more free selenocysteine amino acid residues are located in a light chain. In additional embodiments one or more free selenocysteine amino acid residues are located in a heavy chain. In additional embodiments one or more free selenocysteine amino acid residues are located in a both the heavy and light chain.

In certain embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have been modified so as to not elicit a deleterious immune response in the animal to be treated, e.g., in a human. In one embodiment, the antibody is modified to reduce immunogenicity using art-recognized techniques. For example, antibody components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be humanized, primatized, deimmunized, or chimerized. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into human frameworks and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but “cloaking” them with human-like sections by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, each of which is herein incorporated by reference in its entirety.

De-immunization can also be used to decrease the immunogenicity of an MRD-containing antibody. As used herein, the term “de-immunization” includes alteration of an MRD-containing antibody to modify T cell epitopes (see, e.g., Int. Appl. Pub. WO9852976A1, and WO0034317A2, each if which is herein incorporated by reference in its entirety). For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope “map” is generated from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of antibodies for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

Many different antibody components of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be used in the methods described herein. It is contemplated that catalytic and non-catalytic antibodies can be used in the present invention. For example, Antibody 38C2 is an antibody-secreting hybridoma and has been previously described in Int. Appl. Pub. WO97/21803. 38C2 contains an antibody combining site that catalyzes the aldol addition reaction between an aliphatic donor and an aldehyde acceptor. In a syngeneic mouse model of neuroblastoma, systemic administration of an etoposide prodrug and intra-tumor injection of Ab 38C2 inhibited tumor growth.

The antibody target of the MRD-containing antibody (i.e., the target of the antigenic binding domain) can be any molecule that it is desirable for a MRD-antibody fusion to interact with. For example, the antibody target can be a soluble factor or the antibody target can be a transmembrane protein, such as a cell surface receptor. The antibody target can also be an extracellular component or an intracellular component. In certain embodiments, the antibody target is a factor that regulates cell proliferation, differentiation, or survival. In other embodiments, the antibody target is a cytokine. In another nonexclusive embodiment, the antibody target is a factor that regulates angiogenesis. In another nonexclusive embodiment, the antibody target is a factor that regulates one or more immune responses, such as, autoimmunity, inflammation and immune responses against cancer cells. In another nonexclusive embodiment, the antibody target is a factor that regulates cellular adhesion and/or cell-cell interaction. In certain nonexclusive embodiments, the antibody target is a cell signaling molecule. The ability of an antibody to bind to a target and to block, increase, or interfere with the biological activity of the antibody target can be determined using or routinely modifying assays, bioassays, and/or animal models known in the art for evaluating such activity.

In some embodiments the antibody target of the MRD-containing antibody is a disease-related antigen. The antigen can be an antigen characteristic of a particular cancer, and/or of a particular cell type (e.g., a hyperproliferative cell), and/or of a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leichmaniasis), a fungal infection, a mold, a mycoplasm, a prion antigen, or an antigen associated with a disorder of the immune system.

In some embodiments, the antibody target of the MRD-containing antibody is a target that has been validated in an animal model or clinical setting.

In other embodiments, the antibody target of the MRD-containing antibody is a cancer antigen.

In one embodiment, the antibody target of the MRD-containing antibody is: PDGFRA, PDGFRB, PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D, VEGFR1, VEGFR2, VEGFR3, VEGFC, VEGFD, neuropilin 2 (NRP2), betacellulin, PLGF, RET (rearranged during transfection), TIE1, TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD25, CD32, CD32b, CD44, CD49e (integrin alpha 5), CD55, CD64, CD90 (THY1), CD133 (prominin 1), CD147, CD166, CD200, ALDH1, ESA, SHH, DHH, IHH, patched1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4, WNT4A, WNT5A, WNT5B, WNT7B, WNT8A, WNT10A, WNT10B, WNT16B, LRP5, LRP6, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1 (TNFb, LTa), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF13 (APRIL), TNFSF13B (BLYS), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A), TNFRSF17 (BCMA), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), TNFRSF25 (DR3), ANG1 (ANGPT1), ANG3 (ANGPTL1), ANG4 (ANGPT4), IL1 alpha, IL1 beta, IL1R1, IL1R2, IL2, IL2R, IL5, IL5R, IL6, IL6R, IL8, IL8R, IL10, IL10R, IL12, IL12R, IL13, IL13R, IL15, IL15R, IL18, IL18R, IL19, IL19R, IL21R, IL23, IL23R, mif, XAG1, XAG3, REGIV, FGF1, FGF2, FGF3, FGF4, FGFR1, FGFR2, FGFR3, ALK, ALK1, ALK7, ALCAM, Artemin, Axl, TGFb, TGFb2, TGFb3, TGFBR1, IGFIIR, BMP2, BMP5, BMP6, BMPRI, GDF3, GDF8, GDF9, N-cadherin, E-cadherin, VE-cadherin, NCAM, LICAM (CD171), ganglioside GM2, ganglioside GD2, calcitonin, PSGR, DCC, CDCP1, CXCR2, CXCR7, CCR3, CCR5, CCR7, CCR10, CXCL1, CXCL5, CXCL6, CXCL8, CXCL12, CCL3, CCL4, CCL5, CCL11, Claudin1, Claudin2, Claudin3, Claudin4, TMEFF2, neuregulin, MCSF, CSF, CSFR (fms), GCSF, GCSFR, BCAM, HPV, hCG, SR1F, PSA, FOLR2 (folate receptor beta), BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5, HM1.24, LFA1, LYNX, S100A8, S100A9, SCF, Von Willebrand factor, Lewis Y6 receptor, Lewis Y, CA G250 (CA9), integrin avb3 (CNTO95), integrin avb5, activin B1 alpha, leukotriene B4 receptor (LTB4R), neurotensin NT receptor (NTR), 5T4 oncofetal antigen, Tenascin C, MMP, MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G, cathepsin H, cathepsin L, SULF1, SULF2, MET, UPA, MHC1, MN (CA9), TAG-72, TM4SF1, Heparanase (HPSE), syndecan (SDC1), Ephrin B2, Ephrin B4, or relaxin2. In another embodiment, the antibody target of the MRD-containing antibody is CD137, CD137L, CD152, CD160, CD272, CD273, CD274, CD275 (ICOSL), CD276, CD276 receptor, CD278 (ICOS), CD279, B7-H4, B7H4 receptor, CXCL9, CXCL10, CXCL11, CCL17, CCL21, CCL22, IL35, TNFRSF10b (DR5), or GUC2c (MECIL). An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. The above antibody and MRD targets and those otherwise described herein are intended to be illustrative and not limiting.

In another embodiment, the antibody target of the MRD-containing antibody is CD19, CD22, CD30, CD33, CD38, CD44v6, TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), CD52, CD54 (ICAM), CD74, CD80, CD200 EPCAM (EGP2), neuropilin 1 (NRP1), TEM1, mesothelin, TGFbeta 1, TGFBRII, phosphatidlyserine, folate receptor alpha (FOLR1), TNFRSF10A (TRAIL R1 DR4), TNFRSF10B (TRAIL R2 DR5), CXCR4, CCR4, CCL2, HGF, CRYPTO, VLA5, TNFSF9 (41BB Ligand), TNFRSF9 (41BB), CTLA4, HLA-DR, IL6, TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), MUC1, MUC18, mucin CanAg, ganglioside GD3, EGFL7, PDGFRa, IL21, IGF1, IGF2, CD117 (cKit), PSMA, SLAMF7, carcinoembryonic antigen (CEA), FAP, integrin avb3, or integrin α5β3. In an additional embodiment, the antibody target of the MRD-containing antibody is CD70, LAG3 or KIR. An MRD that binds to one of the above targets are encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention.

In particular embodiments, the antibody of the MRD-containing antibody competes for target binding with an antibody selected from: siplizumab CD2 (e.g., MEDI-507, MedImmune), blinatumomab CD19 CD3 (e.g., MT103, Micromet/MedImmune); XMAB®5574 CD19 (Xencor), SGN-19A CD19 (Seattle Genetics), ASG-5ME (Agenesys and Seattle Genetics), MEDI-551 CD19 (MedImmune), epratuzumab CD22 (e.g., hLL2, Immunomedics/UCB), inotuzumab ozogamicin CD22 (Pfizer), iratumumab CD30 (e.g., SGN-30 (Seattle Genetics) and MDX-060 (Medarex)), XMAB®2513 CD30 (Xencor), brentuximab vedotin CD30 (e.g., SGN-35, Seattle Genetics), gemtuzumab ozogamicin CD33 (e.g., MYLOTARG®, Pfizer), lintuzumab CD33 (e.g., antibody of Seattle Genetics), MOR202, CD38 (MorphoSys), daratumumab CD38 (e.g., Genmab antibody), CP870893 CD40 (Pfizer), dacetuzumab CD40 (e.g., SGN40, Seattle Genetics), ANTOVA® CD40 (Biogen Idec), lucatumumab CD40 HCD122, Novartis) XMAB®5485 CD40 (Xencor), teneliximub, ruplizumab CD40L (e.g., ANTOVA®) bivatuzumab mertansine CD44v6, alemtuzumab CD52 (e.g., CAMPATH®/MABCAMPATH®, Genzyme/Bayer), BI505 ICAM1 (Bioinvent), milatuzumab CD74 (e.g., antibody of Immunomedics), galiximab CD80 (Biogen Idec), BMS663513 4-1BB (Bristol-Myers Squibb), Alexion CD200 antibody (Alexion), edrecolomab EPCAM (e.g., MAb17-1A, PANOREX® (GlaxoSmithKline), AT003 EPCAM (Affitech)), adecatumumab EPCAM (e.g., MT201, Micromet), oportuzumab monatox EPCAM, Genentech anti-NRP1 antibody, MORAB004 TEM1 (Morphotek), MORAB009 mesothelin (Morphotek), lerdelimumab TGFb1 (e.g., CAT-152, Cambridge Antibody Technology), metelimumab TGFb1 (e.g., CAT-192, Cambridge Antibody Technology), ImClone anti-TGFBRII antibody, bavituximab phosphatidylserine (e.g., antibody of Peregrine (Peregrine Pharmaceuticals)), AT004 phosphatidylserine (Affitech), AT005 phosphatidylserine (Affitech), MORAB03 folate receptor alpha (Morphotek), farletuzumab folate receptor alpha cancer (e.g., MORAB003, Morphotek), CS1008 DR4 (Sankyo), mapatumumab DR4 (e.g., HGS-ETR1, Human Genome Sciences), LBY135 DR5 (Novartis), AMG66 DR5 (Amgen), Apomab DR5 (Genentech), PRO95780 (Genentech), lexatumumab DR5 (e.g., HGS-ETR2, Human Genome Sciences), conatumumab DR5 (e.g., AMG655, Amgen), tigatuzumab DR5 (e.g., CS-1008), AT009 CXCR4 (Affitech), AT008 CCR4 (Affitech), CNTO-888 CCL2 (Centocor), AMG102 HGF (Amgen), CRYPTO antibody (Biogen Idec), M200 antibody VLA5 (Biogen Idec), ipilimumab CTLA4 (e.g., MDX-010, Bristol-Myers Squibb/Medarex), belatacept CTLA4 ECD (e.g., CP-675,206, Pfizer), IMMU114 HLA-DR (Immunomedics), apolizumab HLA-DR, toclizumab IL6R (e.g., ACTEMR®A/ROACTREMRA®, Hoffman-La Roche), OX86 OX40, pemtumomab PEM/MUC1 (Theragyn), ABX-MA1 MUC-18 (Abgenix), clivatuzumab MUC-18 (e.g., hPAM4, Immunomedics), cantuzumab mertansine mucin CanAg, ecromeximab (Ludwig Institute), Genentech anti-EGFL7 antibody, AMG820 CSFR (Amgen), olaratumab PDGFRa (e.g., antibody of Imclone (Imclone)), IL21 antibody Zymogenetics (Zymogenetics), MEDI-573 IGF1/IGF2 (MedImmune), AMG191 cKit (Amgen), etaracizumab (e.g., MEDI-522, MedImmune), and MLN591 PSMA (Millennium Pharmaceuticals), elotuzumab SLAMF7 (e.g., HuLuc63, BMS), labetuzumab CEA (CEA-CIDE®, Immunomedics), sibrotuzumab FAP, CNTO95 integrin avb3 (Centocor), VITAXIN® integrin avb3 (MedImmune), and voloximab α5β1 (antibody targets are italicized). MRDs that compete for target binding with one of the above antibodies are encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention.

In additional embodiments, the antibody of the MRD-containing antibody competes for target binding with an antibody selected from: MDX-1342 CD19 (BMS), SGN-CD19A CD19 (Seattle Genetics), an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, ofatumumab CD20 (e.g., ARZERRA®, GENMAB), veltuzumab CD20 (hA20, Takeda and Nycomed), PRO70769 CD20 (Genentech; see e.g., Intl. Appl. No. PCT/US2003/040426), AMG780 Tie2/Ang1 (Amgen), REGN910 ANG2 (Regeneron), and anti-CD22 antibody described in U.S. Pat. No. 5,789,554 (Immunomedics), lumiliximab CD23 (e.g., IDEC152, Biogen), IDEC-152 CD23 (Biogen), MDX-1401 CD30 (BMS), HeFi-1 CD30 (NCI), daratumumab CD38, an anti CD-40 antibody described in Intl. Appl. Publ. No. WO2007124299 (Novartis), IDEC-131 CD40L (Biogen), MDX-1411 CD70 (BMS), SGN-75 CD70 ADC (Seattle Genetics), HuMax-CD74™ CD74 ADC (Genmab), IDEC-114 CD80 (Biogen), TRC105 CD105/endoglin (Tracon), ABX-CBL CD147 (Amgen), RG1HuMax-TF™ Tissue Factor (TF) (Genmab), HuMax-Her2™ ErbB2 (Genmab), Trastuzumab-DM1 ErbB2-DM1 (Genentech), AMG888 HER3 (Amgen and Daiichi Sankyo), HuMV833 VEGF (Tsukuba Research Lab, see, e.g., Intl. Appl. Publ. No. WO/2000/034337), IMC-18F1 VEGFR1 (Imclone), IMC-1C11 VEGFR (Imclone), DC101 VEGFR2 (Imclone), KSB-102 EGFR (KS Biomedix), mAb-806 EGFR (Ludwig Institute for Cancer Research), MR1-1 EGFRvIII toxin (IVAX, National Cancer Institute), HuMax-EGFR EGFR (Genmab, see, e.g., U.S. application Ser. No. 10/172,317), IMC-11F8 EGFR (Imclone), CDX-110 EGFRvIII (AVANT Immunotherapeutics), zalumumab EGFR (Genmab), 425, EMD55900 and EMD62000 EGFR (Merck KGaA, see, e.g., U.S. Pat. No. 5,558,864), ICR62 EGFR (Institute of Cancer Research, see, e.g., Intl. Appl. Publ. No. WO95/20045), SC100 EGFR (Scancell and ISU Chemical), MOR201 FGFR-3 (Morphosys), ARGX-111 c-Met (arGEN-X), HuMax-cMet™ cMet (Genmab), GC-1008 TGEb1 (Genzyme), MDX-070 PMSA (BMS), huJ591 PSMA (Cornell Research Foundation), muJ591 PSMA (Cornell Research Foundation), GC1008 TGFb (Genzyme), NG-1 Ep-CAM (Xoma), MOR101 ICAM-1 (CD54) (Morphosys), MOR102 ICAM-1 (CD54)(Morphosys), ABX-MA1 MUC18 (Abgenix), HumaLYM (Intracel), HumaRAD-HN (Intracel), HumaRAD-OV (Intracel), ARGX-110 and ARGX-111 (arGEN-X), HuMax-Lymphoma (Genmab and Amgen), Milatuzumab CD74 (e.g., IMMU-115, IMMU-110; Immunomedics), HuMax-Cancer Heparanase I (Genmab), Hu3S193 Lewis (y) (Wyeth, Ludwig Institute of Cancer Research), RAV12 N-linked carbohydrate epitope (Raven), nimotuzumab (TheraCIM, hR3; YM Biosciences, see, e.g., U.S. Pat. Nos. 5,891,996 and 6,506,883), BEC2 GD3 (Imclone), ⁹⁰Ytacatuzumab tetraxetan alpha fetoprotein (e.g., FP-CIDE®, Immunomedics), KRN330 (Kirin), huA33 A33 (Ludwig Institute for Cancer Research), mAb 216 B cell glycosylated epitope (NCI), REGN421 DLL4 (Regeneron), ASG-5ME SLC44A4 ADC (AGS-5), ASG-22ME Nectin-4 ADC, CDX-1307 (MDX-1307), hCGb (Celldex), parathyroid hormone-related protein (PTH-rP) (UCB), MT293 cleaved collagen (TRC093/D93, Tracon), KW-2871 GD3 (Kyowa), KIR (1-7F9) KIR (Novo), A27.15 transferrin receptor (Salk Institute, see, e.g., Intl. Appl. Publ. No. WO2005/111082) and E2.3 transferrin receptor (Salk Institute). MRDs that compete for target binding with one of the above antibodies are encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention. In additional embodiments, one of the above-described antibodies is the antibody of the MRD-containing antibody.

In particular embodiments, the antibody of the MRD-containing antibody is an antibody selected from: siplizumab CD2 (e.g., MEDI-507, MedImmune), blinatumomab CD19 CD3 (e.g., MT103, Micromet/MedImmune); XMAB®5574 CD19, (Xencor), SGN-19A CD19 (Seattle Genetics), ASG-5ME (Agenesys and Seattle Genetics), MEDI-551 CD19 (MedImmune), epratuzumab CD22 (e.g., hLL2, Immunomedics/UCB), inotuzumab ozogamicin CD22, iratumumab CD30 (e.g., SGN-30 (Seattle Genetics) and MDX-060 (Medarex)), XMAB®2513 CD30 (Xencor), brentuximab vedotin CD30 (e.g., SGN-35, Seattle Genetics), gemtuzumab ozogamicin CD33 (e.g., MYLOTARG®, Pfizer), lintuzumab CD33 (e.g., antibody of Seattle Genetics), MOR202 CD38 MorphoSys), daratumumab CD33 (e.g., Genmab antibody), CP870893 CD40 (Pfizer), dacetuzumab CD40 (e.g., SGN40, Seattle Genetics), ANTOVA® CD40 (Biogen Idec), lucatumumab CD40 (e.g., FWD 122, Novartis) XMAB®5485 CD40 (Xencor), teneliximab, ruplizumab CD40L (e.g., ANTOVA®), bivatuzumab mertansine CD44v6, alemtuzumab CD52 (e.g., CAMPATH®/MABCAMPATH®, Genzyme/Bayer), BI505 ICAM1 (Bioinvent), milatuzumab CD74 (e.g., antibody of Immunomedics), galiximab CD80 (Biogen Idec), BMS663513 4-1BB (Bristol-Myers Squibb), Alexion CD200 antibody (Alexion), edrecolomab EPCAM (e.g., MAb17-1A, PANOREX® (GlaxoSmithKline), AT003 EPCAM (Affitech)), adecatumumab EPCAM (e.g., MT201, Micromet), oportuzumab monatox EPCAM, Genentech anti-NRP1 antibody, MORAB004 TEM1 (Morphotek), MORAB009 mesothelin (Morphotek), lerdelimumab TGFb1 (e.g., CAT-152, Cambridge Antibody Technology), metelimumab TGFb1 (e.g., CAT-192, Cambridge Antibody Technology), ImClone anti-TGFBRII antibody, bavituximab phosphatidylserine (e.g., antibody of Peregrine (Peregrine Pharmaceuticals)), AT004 phosphatidylserine (Affitech), AT005 phosphatidylserine (Affitech), MORAB03 folate receptor alpha (Morphotek), farletuzumab folate receptor alpha cancer (e.g., MORAB003, Morphotek), CS1008 DR4 (Sankyo), mapatumumab DR4 (e.g., HGS-ETR1, Human Genome Sciences), LBY135 DR5 (Novartis), AMG66 DR5 (Amgen), Apomab DR5 (Genentech), PRO95780 (Genentech), lexatumumab DR5 (e.g., HGS-ETR2, Human Genome Sciences), conatumumab DR5 (e.g., AMG655, Amgen), tigatuzumab (e.g., CS-1008), AT009 CXCR4 (Affitech), AT008 CCR4 (Affitech), CNTO-888 CCL2 (Centocor), AMG102 HGF (Amgen), CRYPTO antibody (Biogen Idec), M200 antibody VLA5 (Biogen Idec), ipilimumab CTLA4 (e.g., MDX-010, Bristol-Myers Squibb/Medarex), belatacept CTLA4 ECD (e.g., CP-675,206, Pfizer), IMMU114 HLA-DR (Immunomedics), apolizumab HLA-DR, toclizumab IL6R (e.g., ACTEMR®A/ROACTREMRA®, Hoffman-La Roche) OX86 OX40, pemtumomab PEM/MUC1 (Theragyn), ABX-MA1 MUC-18 (Abgenix), cantuzumab mertansine mucin. CanAg, ecromeximab (Ludwig Institute), Genentech anti-EGFL7 antibody, AMG820 CSFR (Amgen), olaratumab PDGFRa (e.g., antibody of Imclone (Imclone)), IL21 antibody Zymogenetics (Zymogenetics), MEDI-573 IGF1/IGF2 (MedImmune), AMG191 cKit (Amgen), etaracizumab (e.g., MEDI-522, MedImmune), MLN591 PSMA (Millennium Pharmaceuticals), elotuzumab SLAMF7 (e.g., HuLuc63, PDL), labetuzumab CEA (CEA-CIDE®, Immunomedics), sibrotuzumab FAP, CNTO95 integrin avb3 (Centocor), VITAXIN® integrin avb3 (MedImmune), and voloximab α5β1 (e.g., M200, PDL and Biogen Idec).

In an additional embodiment, the antibody target of the MRD-containing antibody is ALK1. In one embodiment, the antibody is PF-3,446,962 (Pfizer). In another embodiment, the antibody binds to the same epitope as PF-3,446,962. In a further embodiment, the antibody competitively inhibits binding of PF-3,446,962 to ALK1. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for ALK1 binding with PF-3,446,962 are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is CD22. In one embodiment, the antibody is inotuzumab (e.g., inotuzumab ozogamicin CMC-544, PF-5,208,773; Pfizer). In one embodiment, the antibody binds to the same epitope as inotuzumab. In another embodiment, the antibody competitively inhibits binding of inotuzumab to CD22. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD22 binding with inotuzumab are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is CRYPTO. In one embodiment, the antibody is the Biogen CRYPTO antibody that has advanced to phase I clinical trials (Biogen Idec). In another embodiment, the antibody binds to the same epitope as the Biogen CRYPTO antibody. In a further embodiment, the antibody competitively inhibits binding of the Biogen CRYPTO antibody to CRYPTO. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CRYPTO binding with the Biogen CRYPTO antibody are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is TNFSF5 (CD40 LIGAND). In one embodiment, the antibody is the Biogen CD40L antibody that has advanced to phase I clinical trials (Biogen Idec). In another embodiment, the antibody binds to the same epitope as the Biogen CD40L antibody. In a further embodiment, the antibody competitively inhibits binding of the Biogen CD40L antibody to CD40L. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD40L binding with the Biogen CD40L antibody are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is CD80. In one embodiment, the antibody is galiximab (Biogen Idec). In another embodiment, the antibody binds to the same epitope as galiximab. In a further embodiment, the antibody competitively inhibits binding of galiximab to CD80. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD80 binding with galiximab are also encompassed by the invention.

In additional embodiments, an MRD-containing antibody binds CD80 and a target selected from: CD2, CD3, CD4, CD19, CD20, CD22, CD23, CD30, CD33, TNFRSF5 (CD40), CD52, CD74, TNFRSF10A (DR4), TNFRSF10B (DR5), VEGFR1, VEGFR2 and VEGF. In additional embodiments, an MRD-containing antibody binds CD80 and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD80 and also at least bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD80. In further embodiments, the antibody component of the MRD-containing antibody is galiximab. In a further embodiment, the antibody of the MRD-containing antibody binds a molecule on the surface of a cell and MRDs of the MRD-containing antibody binds TNFRSF10B (DR5) through one or more epitopes with a valency of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

In an additional embodiment, the antibody target of the MRD-containing antibody is MCSF. In one embodiment, the antibody is PD-360,324 (Pfizer). In another embodiment, the antibody binds to the same epitope as PD-360,324. In a further embodiment, the antibody competitively inhibits binding of PD-360,324 to MCSF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for MCSF binding with PD-360,324 are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is CD44. In one embodiment, the antibody is PF-3,475,952 (Pfizer). In another embodiment, the antibody binds to the same epitope as PF-3,475,952. In a further embodiment, the antibody competitively inhibits binding of PF-3,475,952 to CD44. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD44 binding with PF-3,475,952 are also encompassed by the invention.

In an additional embodiment, the antibody target of the MRD-containing antibody is p-cadherin (CDH3). In one embodiment, the antibody is PF-3,732,010 (Pfizer). In another embodiment, the antibody binds to the same epitope as PF-3,732,010. In a further embodiment, the antibody competitively inhibits binding of PF-3,732,010 to p-cadherin. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for p-cadherin binding with PF-3,732,010 are also encompassed by the invention.

In another embodiment, the antibody target of the MRD-containing antibody is ANG2 (ANGPT2). In one embodiment, the antibody is MEDI3617 (MedImmune). In one embodiment, the antibody binds to the same epitope as MEDI3617. In another embodiment, the antibody competitively inhibits binding of MEDI3617 to ANG2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for ANG2 binding with MEDI3617 are also encompassed by the invention.

In other embodiments, the antibody component of the MRD-containing antibody is an ANG-2 binding antibody disclosed in U.S. Pat. Nos. 7,063,965, 7,063,840, 6,645,484, 6,627,415, 6,455,035, 6,433,143, 6,376,653, 6,166,185, 5,879,672, 5,814,464, 5,650,490, 5,643,755, 5,521,073; U.S. Appl. Publ. Nos. 2011/0158978 (e.g., H4L4), 2006/0246071, 2006/0057138, 2006/0024297, 2006/0018909, 2005/0100906, 2003/0166858, 2003/0166857, 2003/0124129, 2003/0109677, 2003/0040463 and 2002/0173627; or Intl. Appl. Publ. Nos. WO2006/020706, WO2006/045049, WO2006/068953, or WO2003/030833 (the disclosure of each of which is herein incorporated by reference in its entirety). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for ANG2 binding with these antibodies are also encompassed by the invention.

In another embodiment, an MRD-containing antibody binds ANG2 and additionally binds a target selected from: VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNF, FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL1, IL1beta, IL6, IL8, IL18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind ANG2 and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In further embodiments, the antibody component of the MRD-containing antibody is MEDI3617, AMG780 or REGN910. In further embodiments, the antibody component of the MRD-containing antibody is H4L4.

In particular embodiments, the MRD-containing antibody binds ANG2 and TNF. In additional embodiments, the MRD-containing antibody binds ANG2 and IL6. In other embodiments, the MRD-containing antibody binds ANG2 and ILL In further embodiments, the administered MRD-containing antibody binds ANG2, IL6 and TNF. In further embodiments, the administered MRD-containing antibody binds ANG2, IL1 and TNF. In further embodiments, the MRD-containing antibody binds ANG2, IL1, 1L6 and TNF.

In particular embodiments, the MRD-containing antibody binds ANG2 and TNF and the antibody component of the MRD-containing antibody is adalimumab. In another embodiment, the MRD-containing antibody competes with adalimumab for binding to TNF.

In additional embodiments, the antibody component of the MRD-containing antibody binds ANG2. In further embodiments, the antibody component of the MRD-containing antibody is an ANG2 binding antibody selected from SAITAng-2-1, SAITAng-2-2, SAITAng-2-3, SAITAng-2-4 or another antibody disclosed in Intl. Appl. Publ. No. WO2009/142460. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having an antibody and/or 1, 2, 3, 4, 5, 6, or more MRDs that compete for ANG2 binding with one or more of these antibodies are also encompassed by the invention.

In additional embodiments, the antibody component of the MRD-containing antibody binds TIE2. In further embodiments, the antibody component of the MRD-containing antibody is a TIE2 binding antibody disclosed in U.S. Pat. Nos. 6,365,154 and 6,376,653; U.S. Appl. Publ. Nos. 2007/0025993, 2006/0057138 and 2006/0024297; or Intl. Appl. Publ. Nos. WO2006/020706, WO2000/018437 and WO2000/018804 (the disclosure of each of which is herein incorporated by reference in its entirety). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having an antibody and/or 1, 2, 3, 4, 5, 6, or more MRDs that compete for TIE binding with one or more these antibodies are also encompassed by the invention.

In certain embodiments, the antibody target of the MRD-containing antibody is EGFR(ErbB1), ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-I receptor, prostate specific membrane antigen, an integrin, or cMet.

In one embodiment, the antibody in the MRD-containing antibody specifically binds EGFR(ErbB1). In a specific embodiment, the antibody is ERBITUX® (IMC-C225). In one embodiment, the antibody binds to the same epitope as ERBITUX®. In another embodiment, the antibody competitively inhibits binding of ERBITUX® to EGFR. In another embodiment, the antibody in the MRD-containing antibody inhibits EGFR dimerization. In another specific embodiment, the antibody is matuzumab (e.g., EMD 72000, Merck Serono) or panitumumab (e.g., VECTIBIX®, Amgen). In another embodiment, the antibody binds to the same epitope as matuzumab or panitumumab. In another embodiment, the antibody competitively inhibits binding of matuzumab or panitumumab to EGFR. In another embodiment, the antibody is ABX-EGF (Immunex) or MEDX-214 (Medarex). In another embodiment, the antibody binds to the same epitope as ABX-EGF or MEDX-214. In another embodiment, the antibody competitively inhibits binding of ABX-EGF or MEDX-214 to EGFR. In another specific embodiment, the antibody is zalutumumab (Genmab) or nimotuzumab (Biocon). In an additional embodiment, the antibody binds to the same epitope as zalutumumab (Genmab) or nimotuzumab (Biocon). In another embodiment, the antibody competitively inhibits binding of zalutumumab (Genmab) or nimotuzumab (Biocon) to EGFR.

In one embodiment, an MRD-containing antibody binds EGFR(ErbB1) and a target selected from: HGF, CD64, CDCP1, RON, cMET, ErbB2, ErbB3, IGF1R, PLGF, RGMa, PDGFRa, PDGFRb, VEGFR1, VEGFR2, TNFRSF10A (DR4), TNFRSF10B (DR5), IGF1,2, IGF2, CD3, CD4, NKG2D and tetanus toxoid. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) binds at least 1, 2, 3, 4, 5 or more of these targets. In specific embodiments, the antibody component of the MRD-containing antibody binds EGFR. In further embodiments, the antibody component of the MRD-containing antibody is matuzumab, panitumumab, MEDX-214, or ABX-EGF. In further embodiments, the antibody component of the MRD-containing antibody is nimotuzumab (Biocon) or zalutumumab. In specific embodiments, the antibody component of the MRD-containing antibody is Erbitux®.

In specific embodiments, the MRD containing antibody binds ErbB1 and additionally binds ErbB3. In some embodiments, the antibody component of the MRD-containing antibody binds ErbB1 and an MRD of the MRD-containing antibody binds ErbB3. In a particular embodiment, the antibody component of the MRD-containing antibody is cetuximab. In additional embodiments, the antibody component of the MRD-containing antibody competes for ErbB1-binding with cetuximab. In another embodiment, the antibody in the MRD-containing antibody is an ErbB1-binding antibody selected from: nimotuzumab (Biocon), matuzumab (Merck KGaA), panitumumab (Amgen), zalutumumab (Genmab), MEDX-214, and ABX-EGF. In additional embodiments, the antibody component, MRD component and/or MRD-containing antibody competes for ErbB1-binding with an antibody selected from: nimotuzumab, matuzumab, panitumumab, and zalutumumab. In other embodiments, the antibody component of the MRD-containing antibody binds ErbB3 and an MRD of the MRD-containing antibody binds ErbB1 In additional embodiments, the antibody component of the MRD-containing antibody is an ErbB3-binding antibody selected from MM121 (Merrimack), 8B8 (Genentech), AV203 (Aveo), and AMG888 (Amgen). In additional embodiments, the antibody component, MRD component and/or MRD-containing antibody competes for ErbB3 binding with an antibody selected from MM121, 8B8, AV203, and AMG888.

In one embodiment the MRD-containing antibody specifically binds ErbB2 (Her2). In a specific embodiment, the antibody is trastuzumab (e.g., HERCEPTIN®, Genentech/Roche). In one embodiment, the antibody binds to the same epitope as trastuzumab. In another embodiment, the antibody competitively inhibits binding of trastuzumab to ErbB2. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with at least 1, 2, 3, 4, 5, 6 of the above antibodies.

In other embodiments, the antibody in the MRD-containing antibody specifically binds to ErbB2. In one embodiment, the antibody in the MRD-containing antibody is an antibody that specifically binds to the same epitope as the anti-ErbB2 antibody trastuzumab (e.g., HERCEPTIN®, Genentech). In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits ErbB2-binding by the anti-ErbB2 antibody trastuzumab. In yet another embodiment, the antibody in the MRD-containing antibody is the anti-ErbB2 antibody trastuzumab. In another embodiment, the antibody in the MRD-containing antibody inhibits HER2 dimerization. In another embodiment, the antibody in the MRD-containing antibody inhibits HER2 heterodimerization with HER3 (ErbB3). In a specific embodiment, the antibody is pertuzumab (e.g., OMNITARG® and phrMab2C4, Genentech). In another embodiment, the antibody specifically binds to the same epitope as pertuzumab. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits binding of ErbB2 by pertuzumab. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention. Accordingly, in one embodiment the antibody in the MRD-containing antibody is trastuzumab and 1, 2, 3, 4, 5, 6, or more MRDs in the MRD-containing antibody competitively inhibit binding of ErbB2 by pertuzumab.

In another embodiment, the antibody in the MRD-containing antibody is an ErbB2-binding antibody selected from the group: MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics), MGAH22 (MacroGenics), and pertuzumab (OMNITARG™, 2C4; Genentech). An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, or 4 of the above antibodies are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with at least 1, 2, 3 or 4 of the above antibodies.

In specific embodiments, the MRD containing antibody binds ErbB2 and additionally binds ErbB3. In some embodiments, the antibody component of the MRD-containing antibody binds ErbB2 and an MRD of the MRD-containing antibody binds ErbB3. In a particular embodiment, the antibody component of the MRD-containing antibody is trastuzumab. In additional embodiments, the antibody component, MRD component and/or MRD-containing antibody competes for ErbB2-binding with trastuzumab. In another embodiment, the antibody in the MRD-containing antibody is an ErbB2-binding antibody selected from: MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics), MGAH22 (MacroGenics), and pertuzumab (OMNITARG™). In additional embodiments, the antibody component, MRD component and/or MRD-containing antibody competes for ErbB2-binding with an antibody selected from: MDX-210, tgDCC-E1A, MGAH22, and pertuzumab. In other embodiments, the antibody component of the MRD-containing antibody binds ErbB3 and an MRD of the MRD-containing antibody binds ErbB2.

In some embodiments, the antibody in the MRD-containing antibody comprises the CDRs of the anti-ErbB2 antibody trastuzumab. The CDR, VH, and VL sequences of trastuzumab are provided in Table 1.

TABLE 1 CDR Sequence VL-CDR1 RASQDVNTAVAW (SEQ ID NO: 59) VL-CDR2 SASFLYS (SEQ ID NO: 60) VL-CDR3 QQHYTTPPT (SEQ ID NO: 61) VH-CDR1 GRNIKDTYIH (SEQ ID NO: 62) VH-CDR2 RIYPTNGYTRYADSVKG (SEQ ID NO: 63) VH-CDR3 WGGDGFYAMDY (SEQ ID NO: 64) VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF ATYYCQQHYTTPPTFGQGTKVEIKRT (SEQ ID NO: 65) VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 66)

In one embodiment the MRD-containing antibody specifically binds ErbB3 (Her3). In a specific embodiment, the antibody is MM121 (Merrimack Pharmaceuticals) or AMG888 (Amgen). In one embodiment, the antibody binds to the same epitope as MM121 or AMG888. In another embodiment, the antibody competitively inhibits binding of MM121 or AMG888 to ErbB3. In another specific embodiment, the antibody is AV-203 (AVEO). In one embodiment, the antibody binds to the same epitope as AV-203. In another embodiment, the antibody competitively inhibits binding of AV-203. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1 or both of the above antibodies are also encompassed by the invention

In one embodiment the MRD-containing antibody specifically binds VEGF (VEGFA). In a specific embodiment, the antibody is bevacizumab (e.g., AVASTIN®, Genentech/Roche). In one embodiment, the antibody binds to the same epitope as bevacizumab. In another embodiment, the antibody competitively inhibits binding of bevacizumab to VEGFA. In another embodiment the MRD-containing antibody is AT001 (Affitech). In one embodiment, the antibody binds to the same epitope as AT001. In another embodiment, the antibody competitively inhibits binding of AT001 to VEGFA. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1 or both of the above antibodies are also encompassed by the invention.

In some embodiments, the antibody in the MRD-containing antibody comprises the CDRs of the anti-VEGF antibody bevacizumab. The CDR, VH, and VL sequences of bevacizumab are provided in Table 2.

TABLE 2 CDR Sequence VL-CDR1 SASQDISNYLN (SEQ ID NO: 72) VL-CDR2 FTSSLHS (SEQ ID NO: 73) VL-CDR3 QQYSTVPWT (SEQ ID NO: 74) VH-CDR1 GYTFTNYGMN (SEQ ID NO: 75) VH-CDR2 WINTYTGEPTYAADFKR (SEQ ID NO: 76) VH-CDR3 YPHYYGSSHWYFDV (SEQ ID NO: 77) VL DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGK APKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID NO: 78) VH EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPG KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 79)

In other specific embodiments, the antibody in the MRD-containing antibody specifically binds VEGF. In a specific embodiment, the antibody is bevacizumab (e.g., AVASTIN®, Genentech). In one embodiment, the antibody binds to the same epitope as bevacizumab. In another embodiment, the antibody competitively inhibits binding of bevacizumab to VEGF. In another specific embodiment, the antibody is r84 (Peregrine) or 2C3 (Peregrine). In another embodiment, the antibody binds to the same epitope as r84 or 2C3. In another embodiment, the antibody competitively inhibits VEGF binding by r84 or 2C3. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, or 3 of the above antibodies are also encompassed by the invention.

In one embodiment, an MRD-containing antibody binds VEGF and additionally binds an angiogenic target selected from: VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF, TIE2, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGF and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is r85, 2C3 or AT001. In a specific embodiment, the antibody component of the MRD-containing antibody is bevacizumab.

In one embodiment, an MRD-containing antibody binds VEGF and additionally binds a target selected from: IL1 beta, phosphatidylserine, TNFSF11 (RANKL), TNFSF12 (TWEAK), IGF1,2, IGF2, IGF1, DKK1, SDF2, CXC3CL1 (fractalkine), sclerostin and tetanus toxoid and HGF. In another embodiment, an MRD-containing antibody binds VEGF and additionally binds a target selected from: ErbB3, EGFR, cMet, VEGF, RON (MST1R), DLL4, CDCP1 CD318), NRP1, ROBO4, CD13, CTLA4 (CD152), ICOS (CD278), CD20, CD22, CD30, CD33, CD80 and IL6R. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGF and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is r85, 2C3 or AT001. In a specific embodiment, the antibody component of the MRD-containing antibody is bevacizumab.

In another embodiment, the MRD-containing antibody specifically binds VEGFR1. In one embodiment, the antibody competitively inhibits binding of Aflibercept (Regeneron) to VEGFR1. In another embodiment, the antibody in the MRD-containing antibody inhibits VEGFR1 dimerization. An MRD that competes for target binding with Aflibercept is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with Aflibercept are also encompassed by the invention.

In another embodiment, the MRD-containing antibody specifically binds VEGFR2. In a specific embodiment, the antibody is ramucirumab (e.g., IMC1121B and IMC1C11, ImClone). In another embodiment, the antibody in the MRD-containing antibody inhibits VEGFR2 dimerization. In one embodiment, the antibody binds to the same epitope as ramucirumab. In another embodiment, the antibody competitively inhibits binding of ramucirumab to VEGFR2. In another embodiment, the antibody competitively inhibits binding of Aflibercept to VEGFR2. An MRD that competes for target binding with ramucirumab is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with ramucirumab or Aflibercept are also encompassed by the invention.

In other embodiments, the antibody in the MRD-containing antibody specifically binds to an FGF receptor (e.g., FGFR1, FGFR2, FGFR3, or FGFR4). In one embodiment, the antibody in the MRD-containing antibody is an antibody that specifically binds to FGFR1 (e.g., FGFR1-IIIC). In a specific embodiment, the antibody is IMC-A1 (Imclone). In one embodiment, the antibody binds to the same epitope as IMC-A1. In another embodiment, the antibody competitively inhibits binding of IMC-A1 to FGFR1. In an additional embodiment, the antibody competitively inhibits binding of FP-1039 (Five Prime) to an FGF ligand of FGFR1. In another embodiment, the antibody in the MRD-containing antibody is an antibody that specifically binds to FGFR2 (e.g., FGFR2-IIIB and FGFR2-IIIC). In a further embodiment, the antibody in the MRD-containing antibody is an antibody that specifically binds to FGFR3. In a specific embodiment, the antibody is IMC-A1 (Imclone). In one embodiment, the antibody binds to the same epitope as PRO-001 (ProChon Biotech), R3Mab (Genentech), or 1A6 (Genentech). In another embodiment, the antibody competitively inhibits binding of PRO-001 (ProChon Biotech), R3Mab (Genentech), or 1A6 (Genentech). An MRD that competes for target binding with one of the above antibodies or ligand traps is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1 or more of the above antibodies or ligand traps are also encompassed by the invention.

In one embodiment, the antibody in the MRD-containing antibody specifically binds CD20. In a specific embodiment the antibody is rituximab (e.g., RITUXAN®/MABTHERA®, Genentech/Roche/Biogen Idec). In one embodiment, the antibody binds to the same epitope as rituximab. In another embodiment, the antibody competitively inhibits binding of rituximab to CD20. In an additional embodiment, the antibody is GA101 (Biogen Idec/Roche/Glycart). In one embodiment, the antibody binds to the same epitope as GA101. In another embodiment, the antibody competitively inhibits binding of GA101 to CD20. In an additional embodiment, the antibody is PF-5,230,895 (SBI-087; Pfizer). In one embodiment, the antibody binds to the same epitope as PF-5,230,895. In another embodiment, the antibody competitively inhibits binding of PF-5,230,895 to CD20. In another specific embodiment, the antibody is ocrelizumab (e.g., 2H7; Genentech/Roche/Biogen Idec). In one embodiment, the antibody binds to the same epitope as ocrelizumab. In another embodiment, the antibody competitively inhibits binding of ocrelizumab to CD20. In another specific embodiment, the MRD-containing antibody is selected from: obinutuzumab (e.g., GA101; Biogen Idec/Roche/Glycart), ofatumumab (e.g., ARZERRA® and HuMax-CD20 Genmab), veltuzumab (e.g., IMMU-160, Immunomedics), AME-133 (Applied Molecular Evolution), SGN35 (Millennium), TG-20 (GTC Biotherapeutics), afutuzumab (Hoffman-La Roche) and PRO131921 (Genentech). In another embodiment, the antibody binds to the same epitope as an antibody selected from: obinutuzumab, ofatumumab, veltuzumab, AME-133, SGN35, TG-20 and PRO131921. In another embodiment, the antibody competitively inhibits CD20 binding by an antibody selected from: obinutuzumab, ofatumumab, veltuzumab, AME-133, SGN35, TG-20, afutuzumab, and PRO131921. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with at least 1, 2, 3, 4, 5, 6 of the above antibodies.

In additional embodiments, an MRD-containing antibody binds CD20 and a target selected from: CD19, CD22, CD30, TNFRSF5 (CD40), CD52, CD74, CD80, CD138, VEGFR1, VEGFR2, EGFR, TNFRSF10A (DR4), TNFRSF10B (DR5), TNF, NGF, VEGF, IGF1,2, IGF2, IGF1 and RANKL. In additional embodiments, an MRD-containing antibody binds CD20 and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD20 and also bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD20. In further embodiments, the antibody component of the MRD-containing antibody is an antibody selected from: rituximab, GA101, PF-5,230,895, ocrelizumab obinutuzumab, ofatumumab, veltuzumab, AME-133, SGN35, TG-20, afutuzumab and PRO131921.

In one embodiment the MRD-containing antibody specifically binds IGF1R. In a specific embodiment, the antibody is selected from: cixutumumab (e.g., IMC-A12, Imclone), figitumumab (e.g., CP-751,871, Pfizer), AMG479 (ganitumab, Amgen/Millennium), BIIB022 (Biogen Idec), SCH 717454 (Schering-Plough), and R1507 (Hoffman La-Roche). In one embodiment, the antibody binds to the same epitope as an antibody selected from: cixutumumab, figitumumab, AMG479, BIIB022, SCH 717454, and R1507. In another embodiment, the antibody competitively inhibits IGF1R binding by an antibody selected from: cixutumumab, figitumumab, AMG479, BIIB022, SCH 717454, and R1507. In a specific embodiment, the antibody is figitumumab. In another specific embodiment, the antibody binds to the same epitope as figitumumab. In a further specific embodiment, the antibody competitively inhibits IGF1R binding by figitumumab. In an additional specific embodiment, the antibody is BI1B022. In another specific embodiment, the antibody binds to the same epitope as BIIB022. In a further specific embodiment, the antibody competitively inhibits IGF1R binding by BIIB022. In another embodiment, the antibody in the MRD-containing antibody inhibits IGF1R dimerization. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for IGF1R binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for IGF1R binding with at least 1, 2, 3, 4, 5, 6 of the above antibodies.

In additional embodiments, an MRD-containing antibody binds IGF1R and a target selected from: EGFR, ErbB2, ErbB3, PDGFRa, PDGFRb, cMet, TNFRSF10A (DR4), TNFRSF10B (DR5), CD20, NKG2D, VEGF, PGE2, IGF1, IGF2 and IGF1,2. In additional embodiments, an MRD-containing antibody binds IGF1R and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind IGF1R and bind at 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds IGF1R. In further embodiments, the antibody component of the MRD-containing antibody is selected from: cixutumumab, figitumumab, AMG479, BIIB022, SCH 717454, and R1507.

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds a target (e.g., ligand, receptor, or accessory protein) associated with an endogenous blood brain barrier (BBB) receptor mediated transport system (e.g., the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor mediated transport systems) and is capable of crossing to the brain side of the BBB. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has 2, 3, 4, 5, or more binding sites (i.e., is capable of multivalently binding) a target antigen (e.g., ligand, receptor, or accessory protein) associated with an endogenous BBB receptor mediated transport system (e.g., the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor mediated transport systems). In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has a single binding site for a target associated with an endogenous BBB receptor mediated transport system. In further embodiments, the multivalent and monovalent multispecific composition has 2, 3, 4, 5, or more single binding sites for a target associated with an endogenous BBB receptor mediated transport system. In further embodiments, the MRD-containing antibody binds 1, 2, 3, 4, 5, or more targets located on the brain (cerebrospinal fluid) side of the BBB. In further embodiments, the MRD-containing antibody additionally binds 1, 2, 3, 4, 5, or more targets located on the brain (cerebrospinal fluid) side of the BBB. In particular embodiments, the MRD-containing antibody binds 1, 2, 3, 4, 5, or more targets associated with a neurological disease or disorder. In particular embodiments, the neurological disease or disorder is selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g., HIV-associated dementia). Accordingly, the invention encompasses methods of treating a patient by administering a therapeutically effective amount of a multivalent and monovalent multispecific composition to treat a neurological disease or disorder selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g., HIV-associated dementia). In another embodiment, the multivalent and monovalent multispecific composition is administered to a patient to treat a brain cancer, metastatic cancer of the brain, or primary cancer of the brain. In additional embodiments, the multivalent and monovalent multispecific composition is administered to a patient to treat a neurological tumor such as, a glioma (e.g., a glioblastoma, glioblastoma multiforme (GBM), and astrocytoma), ependymoma, oligodendroglioma, neurofibroma, sarcoma, medulloblastoma, primitive neuroectodermal tumor, pituitary adenoma, neuroblastoma or cancer of the meninges (e.g., meningioma, meningiosarcoma and gliomatosis). In particular embodiments the invention encompasses methods of treating a patient by administering a therapeutically effective amount of a multivalent and monovalent multispecific composition to treat a neurodegenerative disease.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds an endogenous BBB receptor mediated transport system selected from the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor mediated transport systems.

In some embodiments, the multivalent and multispecific composition (e.g., MRD-containing antibody) binds transferrin receptor. In additional embodiments, the MRD-containing antibody binds a target selected from: low-density lipoprotein receptor 1 (LRP-1), a LRP-1 ligand or a functional fragment or variant thereof that binds LRP-1, Low-density lipoprotein receptor 2 (LRP-2), a LRP-2 ligand or a functional fragment or variant thereof that binds LRP-1, a transferrin protein or a functional fragment or variant thereof, insulin receptor, TMEM30A, leptin receptor, IGF receptor, an IGFR ligand or a functional fragment or variant thereof, diphtheria receptor, a diphtheria receptor ligand or a functional fragment or variant thereof, choline transporter, a complex that binds choline receptor, an amino acid transporter (e.g., LAT1/CD98, SLC3A2, and SLC7A5), an amino acid transporter ligand or a functional fragment or variant thereof, RAGE, a RAGE ligand or a functional fragment or variant thereof, SLC2A1 and a SLC2A1 ligand or a functional fragment or variant thereof.

In additional embodiments, the multivalent and multispecific composition (e.g., MRD-containing antibody) binds RAGE. In further embodiments, the multivalent and multispecific composition (e.g., MRD-containing antibody) binds RAGE and a target selected from: Abeta, endothelin1, TNF, IL6, MCSF, an AGE, a S100 member, HMGB1, LPS and TLR2. Multivalent and multispecific compositions that bind RAGE and also bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds RAGE.

In additional embodiments, the multivalent and multispecific composition (e.g., MRD-containing antibody) binds a target antigen associated with an endogenous blood brain barrier (BBB) receptor mediated transport system and also binds a target antigen selected from alpha-synuclein, RGM A, NOGO A, NgR, OMGp MAG, CSPG, neurite inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4) an ephrin, A-beta, AGE (S100 A, amphoterin), NGF, soluble A-B, aggrecan, midkine, neurocan, versican, phosphacan, Te38, and PGE2, IL1, IL1R, IL6, IL6R, IL12, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), CD45RB, CD52, CD200, VEGF, VLA4, TNF alpha, Interferon gamma, GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a and MCP-1. In a further embodiment, the MRD-containing antibody has a single binding site for a target associated with an endogenous blood brain barrier (BBB) receptor mediated transport system and further binds a target selected from alpha-synuclein, RGM A, NOGO A, NgR, OMGp MAG, CSPG, neurite inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4) an ephrin, A-beta, AGE (S100 A, amphoterin), NGF, soluble A-B, aggrecan, midkine, neurocan, versican, phosphacan, Te38, PGE2, IL1R, IL6, IL6R, IL12, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), CD45RB, CD52, CD200, VEGF, VLA4, TNF alpha, Interferon gamma, GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a and MCP-1.

In additional embodiments, the MRD-containing antibody is administered to a patient to treat a neurological disease or disorder selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g., HIV-associated dementia). In one embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for 2 or more of the above targets. In a further embodiment, the multivalent and monovalent multispecific composition contains 2 binding sites for 3 or more targets. In additional embodiments, the targets bound by the multivalent and monovalent multispecific composition are associated with cancer. In a further embodiment the targets bound by the multivalent and monovalent multispecific composition are associated with 1, 2, 3, 4, 5 or more different signaling pathways or modes of action associated with cancer.

In one embodiment, the antibody in the MRD-containing antibody specifically binds integrin. In a specific embodiment, the antibody is selected from: MEDI-522 avb3 (VITAXIN®, MedImmune), CNTO 95 a5b3 (Centocor), JC7U αvβ3, and volociximab a5b1 (e.g., M200, PDL and Biogen Idec). In another embodiment, the antibody binds to the same epitope as an antibody selected from: MEDI-522, CNTO 95, JC7U αvβ3, and volociximab. In another embodiment, the antibody competitively inhibits integrin binding by an antibody selected from: MEDI-522, CNTO 95, JC7U, and M200. In a specific embodiment, the antibody is natalizumab (e.g., TSABRI®, Biogen Idec). In one embodiment, the antibody binds to the same epitope as natalizumab. In another embodiment, the antibody competitively inhibits integrin binding by natalizumab. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention.

In one embodiment, the antibody in the MRD-containing antibody specifically binds cMet. In a specific embodiment, the antibody is selected from: MetMab (OA-5D5, Genentech), AMG-102 (Amgen) and DN30. In another embodiment, the antibody binds to the same epitope as an antibody selected from: MetMab), AMG-102 and DN30. In another embodiment, the antibody competitively inhibits cMET binding by an antibody selected from: MetMab (OA-5D5), AMG-102 and DN30. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, or 3 of the above antibodies are also encompassed by the invention.

In one embodiment, the antibody in the MRD-containing antibody specifically binds cMet and the antibody is selected from: 11E1, CE-355621, LA480 and LMH87. In another embodiment, the antibody binds to the same epitope as an antibody selected from: MetMab), AMG-102 and DN30. In another embodiment, the antibody competitively inhibits cMET binding by an antibody selected from: 11E1, CE-355621, LA480 and LMH87. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3 or 4 of the above antibodies are also encompassed by the invention.

In additional embodiments, an MRD-containing antibody binds cMET and a target selected from: ErbB2, ErbB3, EGFR, IGF1R, NRP1, RON, PDGFRa, PDGFRb, VEGF, VEGFR1, VEGFR2, TGF beta, TGF beta R2, CD82, CD152, NGF, BMP2, BMP4, BMP5, BMP9, BMP10, BMPR-IA, ALK1, a3b1 integrin and HGF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind cMET and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds cMET. In further embodiments, the antibody component of the MRD-containing antibody is an antibody selected from: MetMab, AMG-102 and DN30. In other embodiments, the antibody component of the MRD-containing antibody is an antibody selected from: 11E1, CE-355621, LA480 and LMH87.

In additional embodiments, an MRD-containing antibody binds MST1R (RON). In a specific embodiment, an MRD-containing antibody binds RON and a target selected from: EGFR, ErbB2, ErbB3, VEGFR1, VEGFR2, cMET, CXCR4, VEGF, MST, MTSP1, CDCP1, EPHB2, NGF, CXCL12 and HGF (SF). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind MST1R and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds MST1R.

In one embodiment, the antibody in the MRD-containing antibody specifically binds HGF (SF). In a specific embodiment, the antibody is AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). In another embodiment, the antibody binds to the same epitope as AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). In another embodiment, the antibody competitively inhibits HGF binding by AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). An MRD that competes for target binding with AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO) is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, or 3 of the above antibodies are also encompassed by the invention.

In a specific embodiment, an MRD-containing antibody binds HGF and a target selected from: ErbB2, ErbB3, EGFR, IGF1R, NRP1, RON, PDGFRa, PDGFRb, VEGF, VEGFR1, VEGFR2, TGF beta, TGF beta R2, CD82, CD152, NGF, BMP2, BMP4, BMP5, BMP9, BMP10, BMPR-IA, ALK1, a3b1 integrin, cMET, MST1R (RON), CXCR4, MST, MTSP1, CDCP1, EPHB2, NGF, CXCL12 NRP1 and phosphatidylserine. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind HGF and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds HGF. In further embodiments, the antibody component of the MRD-containing antibody is AMG-102 or SCH 900105.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds a5b1 integrin (VLA5). In a specific embodiment, the antibody is volociximab (e.g., M200 Biogen Idec). In another embodiment, the antibody binds to the same epitope as volociximab. In a further embodiment, the antibody competitively inhibits a5b1 integrin binding by volociximab. An MRD that competes for a5b1 integrin binding with volociximab is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for a5b1 integrin binding with volociximab are also encompassed by the invention.

In another embodiment, the antibody target of the MRD-containing antibody is an antigen associated with an autoimmune disorder, inflammatory or other disorder of the immune system or is associated with regulating an immune response.

In another embodiment the MRD-containing antibody improves the performance of antigen presenting cells (e.g., dendritic cells). In one embodiment the antibody target of the MRD-containing antibody is a member selecting from: CD19, CD20, CD21, CD22, CD23, CD27, CD28, CD30, CD30L, TNFSF14 (LIGHT, HVEM Ligand), CD70, ICOS, ICOSL (B7-H2), CTLA4, PD-1, PDL1 (B7-H1), B7-H4, B7-H3, PDL2 (B7-DC), BTLA, CD46, CD80 (B7-1), CD86 (B7-2), HLA-DR, CD74, PD1, TNFRSF4 (OX40), TNFRSF9 (41BB), TNFSF4 (OX40 Ligand), TNFSF9 (41BB Ligand), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), TNFRSF18 (GITR), MHC-1, TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), FcgammaRIIB, and IL4R.

In one embodiment the antibody target of the MRD-containing antibody is an immunoinhibitory target selected from: IL1, IL1 beta, IL1Ra, L-5, IL6, IL6R, CD26L, CD28, CD80, FcRn, and Fc Gamma RIIB. An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention.

In one embodiment, an MRD-containing antibody binds prostaglandin E2 (PGE2). In a specific embodiment, an MRD-containing antibody binds IL6R and a target selected from: EGFR, IGF1R, IL6R, TNF, NGF, IL1 beta, IL6, IL17A, VEGF, IL15, IL18, S1P and Abeta. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind PGE2 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds PGE2.

In an additional embodiment, the MRD-containing antibody modulates immune responses, immune cell homeostasis, or maintenance of immune tolerance through multi-specific and multi-valent binding of soluble, cell surface or extra-cellular matrix associated molecules.

In one embodiment, the MRD-containing antibody binds one or more cytokines or chemokines that modulate immune responses, immune cell homeostasis, or maintenance of immune tolerance. In a further embodiment, the MRD-containing antibody binds one or more cytokines or chemokines selected from: IL1, IL2, IL6, IL7, IL9, IL10, IL12, IL15, IL17, IL18, IL23, IL35, IFNgamma, IFNalpha, IFNbeta, CCL17, CCL21, CCL22, CXCL9, CXCL10, CXCL11, TGFbeta, LIGHT, and TL1a. In one embodiment, the MRD-containing antibody binds a chemokine that modulates immune responses through immune cell migration. In a further embodiment, the MRD-containing antibody binds a chemokine selected from CCL17, CCL22 and CCL21. In another embodiment, the MRD-containing antibody binds a combination of soluble factors that modulate the activity of T lymphocytes. In a further embodiment, the MRD-containing antibody binds a combination of soluble factors selected from: IL2 and IFNgamma; IL2 and IFNalpha; IL2 and TGFbeta1; IFNgamma and TGFbeta1; and IFNalpha and TGFbeta1. In an additional embodiment, the MRD-containing antibody binds a soluble factor that modulates CD25 expression or the intracellular expression of FOXP3 in T lymphocytes. In some embodiments, the MRD-containing antibody binds two, three, four, five or six cytokines or chemokines that modulate immune responses, immune cell homeostasis, or maintenance of immune tolerance factors.

In an additional embodiment, the MRD-containing antibody binds one or more cell surface associated molecules that modulate immune responses, immune cell homeostasis, or maintenance of immune tolerance. In a further embodiment, the MRD-containing antibody binds one or more cell surface associated molecules selected from: PDL1, PDL2, PD1, CD80, CD86, CTLA4, B7-H2, ICOS, B7-H3, B7-H4, HVEM, BTLA, MHC class1, MHC class II, KIR, TCRalpha, TCRbeta, TCRgamma, LAG3, CD137, C137L, OX40, OX40L, CD70, CD27, CD40, CD40L, GAL9, TIM3, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and A2aR. In one embodiment, the MRD-containing antibody binds one or more cell surface molecules that influence immune cell migration. In a further embodiment, the MRD-containing antibody binds a molecule selected from CCR4, CCR7, and CXCR3. In another embodiment, the MRD-containing antibody binds a combination of cell surface molecules that modulate T lymphocyte function including. In a further embodiment, the MRD-containing antibody binds a combination of soluble factors selected from: CD25 and CTLA4; PD1 and CTLA4; B7-H3 and B7-H3R; B7-H4 and B7H4R; BTLA and HVEM; KIR and LAG3; A2aR and TIM3; TIM3 and CTLA4; PDL1 and PD1; PDL2 and PD1; and CD80 and CD86. In another embodiment, the MRD-containing antibody antagonizes two or more of the inhibitory signals delivered to T lymphocytes through inhibitory receptors. In a further embodiment, the MRD-containing antibody antagonizes two or more inhibitory signals delivered to T lymphocytes including a signal delivered through an inhibitory receptor selected from: PD1, CTLA4, BTLA, HVEM, KIR, LAG3, TIM3, and A2aR. In another embodiment, the MRD-containing antibody enhances the co-stimulatory signals delivered to T lymphocytes through one or more of the co-stimulatory receptors. In a further embodiment, the MRD-containing antibody enhances two or more co-stimulatory signals delivered to T lymphocytes including a signal delivered through a co-stimulatory receptor selected from: CD28, ICOS, OX40 and CD27. In one embodiment, the MRD-containing antibody binds a molecule that modulates CD25 or FOXP3 expression in T lymphocytes. In another embodiment, the MRD-containing antibody binds a molecule that modulates CD39 expression on T lymphocytes. In some embodiments, the MRD-containing antibody binds two, three, four, five or six cell surface associated molecules that modulate immune responses, immune cell homeostasis, or maintenance of immune tolerance.

In an additional embodiment, the MRD-containing antibody binds one or more cell surface molecules expressed on antigen-presenting cells. In a further embodiment, the MRD-containing antibody binds one or more cell surface molecules selected from PDL1, PDL2, CD80, CD86, B7RP1, B7-H3, B7-H4, HVEM, CD137L, OX40, CD70, and GAL3. In one embodiment, the MRD-containing antibody binds a molecule on antigen-presenting cells that modulates CD25 or FOXP3 expression in human T lymphocytes. In another embodiment, the MRD-containing antibody binds a combination of molecules on the surface of an accessory cell. In a further embodiment, the MRD-containing antibody binds a combination of molecules selected from: CD80 and CD86; B7-H3 and B7-H4; PDL1 and PDL2; HVEM and B7-H2; CD137L and HVEM; GAL9 and OX40L; HVEM, PDL1 and PDL2; CD80, CD86 and HVEM; and B7-H3, B7-H4 and B7-H2. The MRD-containing antibody may bind one, two, three, four, five six, seven, eight or more cell surface molecules expressed on antigen-presenting cells.

In another embodiment, the MRD-containing antibody binds one or more cell-surface molecules on the surface of antigen-presenting cells and one or more cell surface molecules expressed on the surface of T lymphocytes. The MRD-containing antibody may bind a combination of one, two, three, or four cell surface molecules expressed on the surface of human antigen-presenting cells and one, two, three, or four cell surface molecules expressed on the surface of human T lymphocytes. In a preferred embodiment, the combination of molecules bound by the MRD-containing antibody share co-stimulatory or inhibitory effects on T lymphocytes. In another preferred embodiment, the combination of molecules bound by the MRD-containing antibody, stabilize cell-cell contact between immune cells.

In an additional embodiment, the MRD-containing antibody antagonizes regulatory T lymphocyte (Treg) function through antagonism of soluble molecules and/or cell surface receptors. In one embodiment, the MRD-containing antibody binds one or more soluble molecules and/or one or more cell surface receptors that coordinately enhance Treg function. In one embodiment, the MRD-containing antibody binds Treg cells and inhibits their function. In another embodiment, the MRD-containing antibody binds Treg cells and enhances their function. In a further embodiment, the MRD-containing antibody binds one or more molecules selected from: PDL1, PDL2, PD1, CD80, CD86, CTLA4, B7-H2, ICOS, B7-H3, B7-H4, HVEM, BTLA, MHC class1, MHC class II, KIR, TCRalpha, TCRbeta, TCRgamma, LAG3, CD137, C137L, OX40, OX40L, CD70, CD27, CD40, CD40L, GAL9, TIM3, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and A2aR. In an additional embodiment, the MRD-containing antibody binds Treg cells and antagonizes the activity of a soluble factor that acts on Treg cells.

The multivalent and multispecific binding properties of MRD-containing antibodies of the invention allow for the rational design of therapeutics that preferentially direct the in vivo localization of the MRD-containing antibody to specific anatomical sites in which the target of the antibody and/or MRD components of the MRD-containing antibodies is present. In one embodiment, the MRD-containing antibody localizes to a site of inflammation by binding of the antibody combining site to a molecule characterized by increased expression in the site of inflammation. In further embodiments, the antibody combining site binds a molecule selected from: an adhesion molecule, an integrin, madCam, CD44 and CD45. In further embodiments, the MRD-containing antibody further comprises a MRD that binds to a cell surface molecule on a Treg that enhances Treg activity. In a further embodiment, the MRD-containing antibody binds a co-stimulatory receptor selected from: CD28, ICOS, OX40 and CD27. In particular embodiments the binding affinity of the MRD-containing antibody for the target in the site of inflammation in vivo is at least 3×, at least 10×, at least 30× or at least 100× that of the MRD-containing antibody for the Treg cell surface molecule.

In another embodiment, the invention provides for a method of preferentially directing the in vivo localization of an MRD-containing antibody to a tumor site. In some embodiments, the antibody combining site of the MRD-containing antibody binds a tumor-associated antigen and an MRD of the MRD-containing antibody binds a cell surface molecule on a Treg that suppresses Treg function. In a further embodiment, the MRD-containing antibody binds a co-stimulatory receptor selected from: PD1, CTLA4, BTLA, HVEM, KIR, LAG3, TIM3, and A2aR. In another embodiment, the antibody combining site of the MRD-containing antibody binds a tumor-associated antigen and an MRD binds a cell surface molecule on a Treg that suppresses Treg function. In an additional embodiment, the antibody combining site binds a tumor associated antigen and an MRD of the MRD-containing antibody binds a cell surface molecule on effector T lymphocytes that enhances the activity of T effector cells. In a further embodiment, the MRD-containing antibody binds a co-stimulatory receptor selected from: CD28, ICOS, OX40 and CD27. In particular embodiments the binding affinity of the MRD-containing antibody for the tumor associated antigen in vivo is at least 3×, at least 10×, at least 30× or at least 100× that of the MRD-containing antibody for the Treg cell surface molecule.

In a further embodiment, the antibody combining site and/or an MRD of an MRD-containing antibody binds a tumor-associated antigen that delivers an inhibitory signal to a T lymphocyte. In particular embodiments, MRD-containing antibody binds CD80 or CD86. In further embodiments, MRD-containing antibody binds B7H1, B7H3 or B7H4.

In another embodiment the antibody target of the MRD-containing antibody is an immunostimulatory target (e.g., an agonist of a target associated immune cell activation (such as, TNFRSF9 (41 BB) or TNFRSF5 (CD40)) or an antagonist of an inhibitory immune checkpoint (such as CTLA-4)). In one embodiment, the antibody target of the MRD-containing antibody is an immunostimulatory target selected from: CD25, CD28, CTLA-4, PD1, PDL1, B7-H1, B7-H4, IL10, TGFbeta, TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM), TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF18 (GITR Ligand), and TNFRSF18 (GITR). In another embodiment, the antibody target of the MRD-containing antibody is an immunostimulatory target selected from: CD272 (BTLA), TIM3, GALS, B7-DC (PDL2), and PDL2 receptor. An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. In specific embodiments, the MRD-containing antibody binds 2, 3 or all 4 targets selected from CTLA-4, TNFRSF18 (GITR), 4-1BB, and TNFRSF5 (CD40). In one embodiment, the MRD-containing antibody binds CTLA-4 and TNFRSF9 (41BB). In another embodiment, the MRD-containing antibody binds CTLA-4 and TNFRSF18 (GITR). In another embodiment, the MRD-containing antibody binds CTLA-4 and TNFRSF5 (CD40). In another embodiment, the MRD-containing antibody binds TNFRSF5 (CD40) and TNFRSF9 (41BB). In another embodiment, the MRD-containing antibody binds TNFRSF4 (OX40) and TNFRSF9 (41BB). In another embodiment, the MRD-containing antibody binds PD1 and B7-H1. In an additional embodiment the MRD-containing antibody enhances an immune response, such as the immune system's anti-tumor response or an immune response to a vaccine.

In another embodiment the antibody target of the MRD-containing antibody is a cytokine selected from: IL1 alpha, IL1 beta, IL18, TNFSF2 (TNFa), LTalpha, LT beta, TNFSF11 (RANKL), TNFSF13B (BLYS), TNFSF13 (APRIL), IL6, IL7, IL10, IL12, IL15, IL17A, IL23, OncoStatinM, TGFbeta, BMP2-15, PDGF (e.g., PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D), an FGF family member (e.g., FGF1, FGF2, FGF4, FGF7, FGF8b and FGF19), VEGF (e.g., VEGFA and VEGFB), MIF, and a type I interferon. MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at least 1, 2, 3, 4, 5, 6 of the above targets.

In another embodiment the antibody target of the MRD-containing antibody is a cytokine selected from: TNF, CD25, CD28, CTLA-4, PD1, PDL1, B7-H1, B7-H4, IL10, TGFbeta, TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM), TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF18 (GITR Ligand), and TNFRSF18 (GITR). An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at least 1, 2, 3, 4, 5, 6 of the above targets.

In one embodiment the antibody target of the MRD-containing antibody is IL1Ra, IL1Rb, IL2, IL3, IL7, IL10, IL11, IL15, IL16, IL17, IL17A, IL17F, IL18, IL19, IL25, IL32, IL33, interferon beta, SCF, BCA1/CXCL13, CXCL1, CXCL2, CXCL6, CXCL13, CXCL16, C3AR, C5AR, CXCR1, CXCR2, CXCR3 CCR1, CCR3, CCR4, CCR7, CCR8, CCR9, CCR10, ChemR23, CCL3, CCL5, CCL11, CCL13, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24, CCL25, CCL26, CCL27, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TREM1, TREM2, FcRn, FcGamma RIIB, oncostatin M, lymphotoxin alpha (LTa), integrin beta 7 subunit, CD49a (integrin alpha 1), integrin a5b3, MIF, ESM1, WIF1, cathepsin B, cathepsin D, cathepsin K, cathepsin S, TNFSF2 (TNFa), TNFSF3 (LTb), TNFRSF3 (LTBR), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM), TNFRSF16 (NGFR), TNFSF18 (GITR Ligand), TNFRSF18 (GITR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), CD14, CD23 CD25, CD28, CD36, CD36L, CD39, CD52, CD91, CD153, CD164, CD200, CD200R, BTLA, CD80 (B7-1), CD86 (B7-2), B7h, ICOS, ICOSL (B7-H2), MHC, CD, B7-H3, B7H4, B7x, SLAM, KIM-1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6, or SLAMF7. An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at least 1, 2, 3, 4, 5, 6 of the above targets. The above antibody and MRD targets and those otherwise described herein are intended to be illustrative and not limiting.

In another embodiment, the antibody target of the MRD-containing antibody is TNFSF1A (TNF/TNF-alpha), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27), TNFSF13B (BLYS), TNFSF13 (APRIL), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), IL1 beta, IL1R, IL2R, IL4-Ra, IL5, IL5R, IL6, IL6R, IL9, IL12, IL13, IL14, IL15, IL15R, IL17f, IL17R, IL17Rb, IL17RC, IL20, IL21, IL22RA, IL23, IL23R, IL31, P, TSLPR, interferon alpha, interferon gamma, B7RP-1, cKit, GMCSF, GMCSFR, CTLA-4, CD2, CD3, CD4, CD11a, CD18, CD20, CD22, CD26L, CD30, TNFRSF5 (CD40), CD80, CD86, CXCR3, CXCR4, CCR2, CCR4, CCR5, CCR8, CCL2, CXCL10, PLGF, PD1, B7-DC (PDL2), B7-H1 (PDL1), alpha4 integrin, A4B7 integrin, C5, RhD, IgE, or Rh. An MRD that binds to one of the above targets is encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at least 1, 2, 3, 4, 5, 6 of the above targets.

In particular embodiments, the antibody target of the MRD-containing antibody competes for target binding with: SGN-70 CD70 (Seattle Genetics), SGN-75 CD70 (Seattle Genetics), Belimumab BLYS (e.g., BENLYSTA®, Human Genome Sciences/GlaxoSmithKline), Atacicept BLYS/APRIL (Merck/Serono), TWEAK (e.g., Biogen mAb), TL1A antibodies of CoGenesys/Teva (e.g., hum11D8, hum25B9, and hum1B4 (U.S. Appl. Publ. No. 2009/0280116), OX40 mAb, humAb OX40L (Genentech), rilonacept IL1 trap (e.g., ARCALYST®, Regeneron), catumaxomab IL1beta (e.g., REMOVAB®, Fresenius Biotech GmbH), Xoma052 IL1beta (Lilly), canakinumab IL1beta (e.g., ILARIS® (Novartis) and ACZ885 (Novartis)), AMG108 IL1R (Amgen), daclizumab IL2Ra (e.g., ZENAPAX®, Hoffman-La Roche), basiliximab IL2Ra (e.g., SIMULECT®, Novartis), AMGN-317 IL4a (Amgen), pascolizumab IL4 (PDL), mepolizumab IL5 (e.g., BOSATRIA®, GlaxoSmithKline), reslizumab IL5 (e.g., SCH55700, Ception Therapeutics), benralizumab IL5R (e.g., MEDI-563 MedImmune), BIW-8405, IL5R (BioWa), etanercept TNFR2-fc (e.g., ENBREL®, Amgen), siltuximab IL6 (e.g., CNT0328, Centocor), CNTO136 IL6 (Centocor), CDP-6038 IL6 (UCB), AMGN-220 IL6 (Amgen), REGN-88 IL6R (Regeneron), tocilizumab IL6R (e.g., ACTEMRA™/ROACTEMRA™, Chugai/Roche), MEDI-528 IL9 (MedImmune), briakinumab IL12/13 (e.g., ABT-874, Abbott), ustekinumab IL12, IL23 (e.g., STELARA® and CNTO 1275, Centocor), TNX-650 IL13 (Tanox), lebrikizumab IL13 (Genentech), tralokinumab IL13 (e.g., CAT354, e.g., Cambridge Antibody Technology), AMG714 IL15 (Amgen), CRB-15 IL15R (Hoffman La-Roche), AMG827 IL17R (Amgen), IL17RC antibody of Zymogenetics/Merck Serono, IL20 antibody of Zymogenetics, IL20 antibody of Novo Nordisk, IL21 antibody of Novo Nordisk (e.g., NCT01038674), IL21 antibody Zymogenetics (Zymogenetics), IL22RA antibody of Zymogenetics, IL31 antibody of Zymogenetics, AMG157 TSLP (Amgen), MEDI-545 interferon alpha (MedImmune), MEDI-546 interferon alpha receptor (MedImmune), AMG811 interferon gamma (Amgen), INNO202 interferon gamma (Innogenetics/Advanced Biotherapy), HuZAF interferon-gamma (PDL), AMG557 B7RP1 (Amgen), AMG191 cKit (Amgen), MOR103 GMCSF (MorphoSys), mavrilimumab GMCSFR (e.g., CAM-3001, MedImmune), tremelimumab CTLA4 (e.g., CP-675,206, Pfizer), iplimumab CTLA4 (e.g., MDX-010, BMS/Medarex), alefacept CD2 (e.g., AMEVIVE®, Astellas), siplizumab CD2 (e.g., MEDI-507, MedImmune), otelixizumab CD3 (e.g., TRX4, Tolerx/GlaxoSmithKline), teplizumab CD3 (e.g., MGA031, MacroGenics/Eli Lilly), visilizumab CD3 (e.g., NUVION®, PDL), muromonab-CD3 CD3 (Ortho), ibalizumab (e.g., TMB-355 and TNX-355, TaiMed Biologics), zanolimumab CD4 (e.g., HUMAX-CD4®, Genmab), cedelizumab CD4 (Euroasian Chemicals), keliximab CD4, priliximab CD4 (e.g., cMT412, Centocor), BT-061 CD4 (BioTest AG), efalizumab CD11a (e.g., RAPTIVA®/XANELIM™, Genentech/Roche/Merck-Serono), MLN01 CD18 (Millennium Pharmaceuticals), epratuzumab CD22 (e.g., Amgen antibody) and hLL2; (Immunomedics/UCB)), aselizumab CD26L, iratumumab CD30 (e.g., SGN30 (Seattle Genetics) and MDX-060 (Medarex), SGN40 CD40 (Seattle Genetics), ANTOVA® CD40 ligand (Biogen Idec), abatacept CD80 CD86 (e.g., ORENCIA®, Bristol-Myers Squibb), CT-011 PD1 (Cure Tech), GITR (e.g., TRX518, (Tolerx), AT010 CXCR3 (Affitech), MLN1202 CCR2 (Millennium Pharmaceuticals), AMG-761 CCR4 (Amgen), HGS004 CCR5 (Human Genome Sciences), PRO 140 (Progenies), MDX-1338 CXCR4 (Medarex), CNTO-888 CCL2 (Centocor), ABN912 CCL2 (Novartis), MDX-1100 CXCL10 (Medarex), TB-403 PLGF (BioInvent), natalizumab integrin Alpha4 subunit (e.g., TYSABRI®, Biogen Idec/Elan), vedolizumab integrin A4B7 (e.g., MLN2, Millennium Pharmaceuticals/Takeda), eculizumab C5 Compliment (e.g., SOLIRIS®, Alexion), pexelizumab C5 Compliment (Alexion), omalizumab IgE (e.g., XOLAIR®, Genentech/Roche/Novartis), talizumab (e.g., TNX-901, Tanox), toralizumab (IDEC 131, IDEC), bertilimumab eotaxin (e.g., iCo-008, iCos Therapeutics Inc.), ozrolimupab RhD (e.g., Sym001, Symphogen A/S), atorolimumab or morolimumab (Rh factor). An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with at least 1, 2, 3, 4, 5 or 6 of the above antibodies.

In particular embodiments, the antibody of the MRD-containing antibody is: SGN-70 CD70 (Seattle Genetics), SGN-75 CD70 (Seattle Genetics), Belimumab BLYS (e.g., BENLYSTA®, Human Genome Sciences/GlaxoSmithKline), BIIB023 TWEAK (Biogen Idec), TL1A antibodies of CoGenesys/Teva (e.g., 11 D8, 25B9, and 1B4 (U.S. Appl. Publ. No. 2009/0280116), OX40 mAb, humAb OX40L (Genentech), catumaxomab IL1beta (e.g., REMOVAB®, Fresenius Biotech GmbH), canakinumab IL1beta (e.g., ILARIS® (Novartis) and ACZ885 (Novartis)), AMG108 IL1R (Amgen), daclizumab IL2Ra (e.g., ZENAPAX®, Hoffman-La Roche), basiliximab IL2Ra (e.g., SIMULECT®, Novartis), AMGN-317 IL4a (Amgen), pascolizumab IL4 (PDL), mepolizumab IL5 (e.g., BOSATRIA®, GlaxoSmithKline), reslizumab IL5 (e.g., SCH55700, Ception Therapeutics), benralizumab IL5R (e.g., MEDI-563, MedImmune), BIW-8405, IL5R (BioWa), siltuximab IL6 (e.g., CNT0328, Centocor), CNTO-136 IL6 (Centocor), CDP-6038 IL6 (UCB), AMGN-220 IL6 (Amgen), REGN-88 IL6R (Regeneron), tocilizumab IL6R (e.g., ACTEMRA™/ROACTEMRA™, Chugai/Roche), MEDI-528 IL9 (MedImmune), briakinumab IL12/13 (e.g., ABT-874, Abbott), ustekinumab IL12, IL23 (e.g., CNTO 1275, Centocor), lebrikizumab IL13 (Genentech), TNX-650 IL13 (Tanox), CAT354 IL13 (Cambridge Antibody Technology), AMG714 IL15 (Amgen), CRB-15 IL15R (Hoffman La-Roche), AMG827 IL17R (Amgen), IL17RC antibody of Zymogenetics/Merck Serono, IL20 antibody of Zymogenetics, IL20 antibody of Novo Nordisk, IL21 antibody of Novo Nordisk, IL21 antibody Zymogenetics (Zymogenetics), IL22RA antibody of Zymogenetics, IL31 antibody of Zymogenetics, AMG157 TSLP (Amgen), MEDI-545 interferon alpha (MedImmune), MEDI-546 interferon alpha receptor (MedImmune), AMG811 interferon gamma (Amgen), INNO202 interferon gamma (Innogenetics/Advanced Biotherapy), HuZAF interferon-gamma (PDL), AMG557B7RP1 (Amgen), AMG191 cKit (Amgen), MOR103 GMCSF (MorphoSys), CAM-3001 GMCSFR (MedImmune), tremelimumab CTLA4 (e.g., CP-675,206, Pfizer), iplimumab CTLA4 (e.g., MDX-010, BMS/Medarex), siplizumab CD2 (e.g., MEDI-507, MedImmune), otelixizumab CD3 (e.g., TRX4, Tolerx/GlaxoSmithKline), muromonab-CD3 CD3 (Ortho), teplizumab CD3 (e.g., MGA031, MacroGenics/Eli Lilly), visilizumab CD3 (e.g., NUVION®, PDL), zanolimumab CD4 (e.g., HUMAX-CD4®, Genmab), cedelizumab CD4 (Euroasian Chemicals), keliximab CD4, priliximab CD4 (e.g., cMT412, Centocor), BT-061 CD4 (BioTest AG), ibalizumab (e.g., TMB-355 and TNX-355, TaiMed Biologics), efalizumab CD11a (e.g., RAPTIVA®/XANELIM™, Genentech/Roche/Merck-Serono), MLN01 CD18 (Millennium Pharmaceuticals), epratuzumab CD22 (e.g., Amgen antibody) and hLL2 (Immunomedics/UCB)), aselizumab CD26L iratumumab CD30 (e.g., SGN30 (Seattle Genetics) and MDX-060 (Medarex), SGN40 CD40 (Seattle Genetics), ANTOVA® CD40 ligand (Biogen Idec), CT-011 PD1 (Cure Tech), AT010 CXCR3 (Affitech), MLN3897 CCR1 (Millennium Pharmaceuticals), MLN1202 CCR2 (Millennium Pharmaceuticals), AMG-761 CCR4 (Amgen), HGS004 CCR5 (Human Genome Sciences), PRO 140 (Progenies), MDX-1338 CXCR4 (Medarex), CNTO-888 CCL2 (Centocor), ABN912 CCL2 (Novartis), MDX-1100 CXCL10 (Medarex), TB-403 PLGF (BioInvent), natalizumab integrin Alpha4 subunit (e.g., TYSABRI®, Biogen Idec/Elan), vedolizumab integrin A4B7 (e.g., MLN02, Millennium Pharmaceuticals/Takeda), eculizumab C5 Compliment (e.g., SOLIRIS®, Alexion pharmaceuticals), omalizumab IgE (e.g., XOLAIR®, Genentech/Roche/Novartis), talizumab (e.g., TNX-901, Tanox), toralizumab (IDEC 131, IDEC), bertilimumab eotaxin (e.g., iCo-008, iCo Therapeutics Inc.), ozrolimupab RhD (e.g., Sym001, Symphogen A/S), atorolimumab or morolimumab (Rh factor).

In additional embodiments, the antibody target of the MRD-containing antibody competes for target binding with an antibody selected from: oxelumab (e.g., RG4930; Genmab), AMG139 (Amgen), AMG181 (Amgen), CNTO 148 TNF (Medarex), an anti-TNF antibody described in U.S. Pat. No. 6,258,562 (BASF), Humicade® TNF (Celltech), HuM291 CD3 fc receptor (PDL), Mik-beta-1 IL-2Rb (CD122) (Hoffman LaRoche), REGN668 IL-4R (Regeneron), sarilumab IL-6R (e.g., REGN88, Regeneron), HuMax-Inflam IL-8 (e.g., HuMax-Inflam™/MDX-018; Genmab and Medarex), anti-IL-12 and/or anti-IL-12p40 antibody disclosed in U.S. Pat. No. 6,914,128 (Abbott), HuMax-IL15 IL15 (Medarex and Genmab), ABX-IL8 IL8 (Abgenix), an anti-IL-18 antibody disclosed in US Appl. Pub. No. 2005/0147610 (Abbott), hCBE-11 LTBR (Biogen), HuMax-TAC IL-2Ra (CD25) (Genmab, see, e.g., Intl. Appl. Publ. No. WO2004045512, MLN01 Beta2 integrin (Xoma), D3H44 ATF (Genentech), MT203 GMCSF (e.g., namilumab, Micromet and Takeda), IFX1/CaCP29 (InflaRx GmbH), CAT-213 Eotaxin 1 (Cambridge Antibody Technologies), MDX-018 IL-8 (e.g., HuMax-Inflam™; Medarex), REGN846 IL-4R (Regeneron, see, e.g., US Appl. Pub. No. 20100291107), REGN728 (Regeneron), RGN846 (Regeneron), T2-18C3 IL1A (MABp1; XBiotech), RA-18C3 IL1A (XBiotech) and CV-18C3 IL1A (XBiotech). An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, 5, 6, or more of the above antibodies are also encompassed by the invention. Thus, the invention encompasses MRD-containing antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with at least 1, 2, 3, 4, 5, or 6 of the above antibodies.

In additional embodiments, one of the above-described antibodies is the antibody of the MRD-containing antibody.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds CTLA4. In a specific embodiment, the antibody is tremelimumab (e.g., CP-675,206, Pfizer). In another embodiment, the antibody binds to the same epitope as tremelimumab. In a further embodiment, the antibody competitively inhibits binding of tremelimumab to CTLA4. In an additional specific embodiment, the antibody is ipilimumab (e.g., MDX-010, Bristol-Myers Squibb/Medarex). In one embodiment, the antibody binds to the same epitope as ipilimumab. In a further embodiment, the antibody competitively inhibits binding of ipilimumab to CTLA4. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CTLA4 binding with tremelimumab or ipilimumab are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds TNFSF12 (TWEAK). In a specific embodiment, the antibody is the TWEAK antibody of Biogen that has advanced to Phase I clinical trials. In another embodiment, the antibody binds to the same epitope as the Biogen TWEAK antibody. In a further embodiment, the antibody competitively inhibits binding of the Biogen TWEAK antibody to TWEAK. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for TWEAK binding with the Biogen TWEAK antibody are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds IL2Ra (CD25). In a specific embodiment, the antibody is daclizumab (e.g., ZENAPAX®). In another embodiment, the antibody binds to the same epitope as daclizumab. In a further embodiment, the antibody competitively inhibits binding of daclizumab to IL2Ra (CD25). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for IL2Ra (CD25) binding with daclizumab are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds CD40 (TNFRSF5). In a specific embodiment, the antibody is CP-870893 CD40 (Pfizer). In another embodiment, the antibody binds to the same epitope as CP-870893. In a further embodiment, the antibody competitively inhibits binding of CP-870893 to CD40. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD40 binding with CP-870893 are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds Alpha4 integrin. In a specific embodiment, the antibody is natalizumab (e.g., TYSABRI®; Biogen Idec/Elan). In one embodiment, the antibody binds to the same epitope as natalizumab. In a further embodiment, the antibody competitively inhibits binding of natalizumab to Alpha4 integrin. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for Alpha4 integrin binding with natalizumab are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds IL22. In a specific embodiment, the antibody is PF-5,212,367 (ILV-094) (Pfizer). In another embodiment, the antibody binds to the same epitope as PF-5,212,367. In a further embodiment, the antibody competitively inhibits binding of PF-5,212,367 to IL22. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for IL22 binding with PF-5,212,367 are also encompassed by the invention.

In an additional embodiment, the antibody in the MRD-containing antibody specifically binds MAdCAM. In a specific embodiment, the antibody is PF-547,659 (Pfizer). In another embodiment, the antibody binds to the same epitope as PF-547,659. In a further embodiment, the antibody competitively inhibits binding of PF-547,659 to MAdCAM. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for MAdCAM binding with PF-547,659 are also encompassed by the invention.

In one embodiment, the antibody in the MRD-containing antibody specifically binds TNF. In a specific embodiment, the antibody is adalimumab (e.g., HUMIRA®/TRUDEXA®, Abbott). In one embodiment, the antibody binds to the same epitope as adalimumab. In another embodiment, the antibody competitively inhibits binding of adalimumab to TNF. In another specific embodiment, the antibody is ATN-103 (Pfizer). In one embodiment, the antibody binds to the same epitope as ATN-103. In another embodiment, the antibody competitively inhibits binding of ATN-103 to TNF. In another specific embodiment, the antibody is infliximab. In one embodiment, the antibody binds to the same epitope as infliximab. In another embodiment, the antibody competitively inhibits binding of infliximab to TNF. In another specific embodiment, the antibody is selected from: certolizumab (e.g., CIMZIA®, UCB), golimumab (e.g., SIMPONI™, Centocor), and AME-527 (Applied Molecular Evolution). In one embodiment, the antibody binds to the same epitope as certolizumab, golimumab, or AME-527. In another embodiment, the antibody competitively inhibits binding of certolizumab, golimumab, or AME-527, to TNF. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4, or 5, of the above antibodies are also encompassed by the invention.

In some embodiments, the antibody in the MRD-containing antibody comprises the CDRs of the anti-TNF antibody adalimumab. The CDR, VH, and VL sequences of adalimumab are provided in Table 3.

TABLE 3 CDR Sequence VL-CDR1 RASQGIRNYLA (SEQ ID NO: 80) VL-CDR2 AASTLQS (SEQ ID NO: 81) VL-CDR3 QRYNRAPYT (SEQ ID NO: 82) VH-CDR1 DYAMH (SEQ ID NO: 83) VH-CDR2 AITWNSGHIDYADSVEG (SEQ ID NO: 84) VH-CDR3 VSYLSTASSLDY (SEQ ID NO: 85) VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGK APKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVA TYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 86) VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG KGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 87)

In one embodiment, an MRD-containing antibody binds TNF (i.e., TNF alpha) and additionally binds a target selected from: Te38, IL12, IL12p40, IL13, TL15, IL17, IL18, IL1beta, IL23, MIF, PGE2, PGE4, VEGF, TNFSF11 (RANKL), TNFSF13B (BLYS), GP130, CD22 and CTLA-4. In another embodiment, an MRD-containing antibody binds TNF alpha, IL6, and TNFSF13B (BLYS). In another embodiment, an MRD-containing antibody binds TNF alpha and TNFSF12 (TWEAK). In additional embodiments, the MRD-containing antibody binds TNF and TNFSF15 (TL1A). In another embodiment, an MRD-containing antibody binds TNF and additionally binds a target selected from NGF, SOST (sclerostin), LPA, IL17A, DKK, alpha Vbeta3, IL23p19, IL2, IL2RA (CD25), IL6, IL6R, IL12p40, IL6, IL10, IL21, IL22 and CD20 binds TNF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNF alpha and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNF alpha. In further embodiments, the antibody component of the MRD-containing antibody is adalimumab, infliximab certolizumab golimumab, CNTO 148, AME-527 or ATN-103.

In other embodiments, the target of the antibody of the MRD-containing antibody is IL6. In some embodiments, the antibody of the MRD-containing antibody is siltuximab (CNT0328, Centocor), CNTO-136 (Centocor), CDP-6038 (UCB), or AMGN-220 (Amgen). In other embodiments, the antibody of the MRD-containing antibody competes with siltuximab (CNT0328, Centocor), CNTO-136 (Centocor), CDP-6038 (UCB), or AMGN-220 (Amgen) for binding to IL6. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2, or more of the above antibodies are also encompassed by the invention.

In one embodiment, an MRD-containing antibody binds IL6. In a specific embodiment, an MRD-containing antibody binds IL6 and a target selected from: IL1, IL1beta, IL1Ra, IL5, CD8, TNFRSF5 (CD40), PDL1, IL6R, IL17A, TNF, VEGF, TNFSF11 (RANKL) and PGE2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind IL6 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds IL6. In further embodiments, the antibody component of the MRD-containing antibody is siltuximab, CNTO136, CDP-6038 or AMGN-220.

In other embodiments, the target of the antibody of the MRD-containing antibody is IL6R. In some embodiments, the antibody of the MRD-containing antibody is REGN-88 (Regeneron) or tocilizumab (ACTEMRA™/ROACTEMRA™, Chugai/Roche). In other embodiments, the antibody of the MRD-containing antibody competes with siltuximab, REGN-88 (Regeneron) or tocilizumab (ACTEMRA™/ROACTEMRA™, Chugai/Roche) for binding to IL6R. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1 or both of the above antibodies are also encompassed by the invention.

In one embodiment, an MRD-containing antibody binds IL6R. In a specific embodiment, an MRD-containing antibody binds IL6R and a target selected from: CD8, TNFRSF5 (CD40), PDL1, IL6, IL17A, TNF, VEGF, TNFSF11 (RANKL) and PGE2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind IL6R and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds IL6R. In further embodiments, the antibody component of the MRD-containing antibody is REGN-88 or tocilizumab.

In some embodiments, an MRD-containing antibody binds TNFSF15 (TL1A). In further embodiments, the MRD-containing antibody binds TL1A and a target selected from: TNF, IFN alpha, IFN gamma, IL1, IL1beta, IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TL1A and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. These compositions have applications in treating diseases and disorders including inflammatory bowel disease and autoimmune diseases such as rheumatoid arthritis. In specific embodiments, the antibody component of the MRD-containing antibody binds TL1a.

In some embodiments, an MRD-containing antibody binds interferon alpha. In further embodiments, the MRD-containing antibody binds interferon alpha and TNFSF13B (BLYS). In further embodiments, the MRD-containing antibody binds interferon alpha, TNFSF13B (BLYS), and a neutrophil extracellular trap (NET). These compositions have applications in treating diseases and disorders including autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematous. In specific embodiments, the antibody component of the MRD-containing antibody binds interferon alpha.

The multivalent and multispecific compositions of the invention also have applications in treating neurologic diseases or disorders including neurodegenerative diseases, pain and neural injury or trauma. In particular embodiments, the target of the antibody of the MRD-containing antibody is: amyloid beta (Abeta), beta amyloid, complement factor D, PLP, ROBO4, ROBO, GDNF, NGF, LINGO, or myostatin. In specific embodiments, the antibody in the MRD-containing antibody is gantenerumab (e.g., R1450, Hoffman La-Roche), bapineuzumab beta amyloid 9 (Elan and Pfizer), solanezumab beta amyloid 9 (Eli Lilly), tanezumab NGF (e.g., RN624, Pfizer), BIIB033 LINGO (Biogen Idec), PF-3,446,879 myostatin (Pfizer), or stamulumab myostatin (Wyeth). In a particular embodiment, the antibody in the MRD-containing antibody is fulranumab (e.g., AMG-403; Amgen/Millennium. In another embodiment, the antibody specifically binds to the same epitope as gantenerumab, bapineuzumab, solarezumab, tanezumab, the Biogen LINGO antibody, or stamulumab. In a further embodiment, the antibody specifically binds to the same epitope as fulranumab. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits target binding by gantenerumab, bapineuzumab, solarezumab, tanezumab, BIIB033, or stamulumab. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits NGF binding by fulranumab. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In an additional embodiment, the target of the antibody of the MRD-containing antibody is beta amyloid. In a specific embodiment, the antibody in the MRD-containing antibody is RN1219 (PF-4,360,365; Pfizer). In another embodiment, the antibody specifically binds to the same epitope as RN1219. In a further embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits beta amyloid binding by RN1219. An MRD that competes for beta amyloid binding with RN1219 is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for beta amyloid binding with RN1219 are also encompassed by the invention.

In an additional embodiment, the target of the antibody of the MRD-containing antibody is NGF. In a specific embodiment, the antibody in the MRD-containing antibody is tanezumab (e.g., RN624, Pfizer). In another embodiment, the antibody specifically binds to the same epitope as tanezumab. In a further embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits NGF binding by tanezumab. An MRD that competes for NGF binding with tanezumab is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for NGF binding with tanezumab are also encompassed by the invention.

In a specific embodiment, an MRD-containing antibody binds NGF and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3, TNFRSF21 (DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19, CD22, TNFRSF5 (CD40), CD80, cMET, NRP1, TNF, LINGO, HGF, IGF1, IGF1,2, IGF2, NGF, Te38, NogoA, RGM A, MAG, OMGp, NgR, TNFSF12 (TWEAK), PGE2, IL1 beta, Semaphorin 3A and Semaphorin 4. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind NGF and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds NGF. In further embodiments, the antibody component of the MRD-containing antibody is tanezumab. In additional embodiments, the antibody component of the MRD-containing antibody competes for NGF binding with tanezumab. In further embodiments, the antibody component of the MRD-containing antibody is MEDI-578. In additional embodiments, the antibody component of the MRD-containing antibody competes for NGF binding with MEDI-578.

In an additional embodiment, the target of the antibody of the MRD-containing antibody is LINGO (e.g., LINGO1). In a specific embodiment, the antibody in the MRD-containing antibody is BIIB033 (Biogen Idec). In another embodiment, the antibody specifically binds to the same epitope as BIIB033. In a further embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits LINGO binding by BIIB033. An MRD that competes for LINGO binding with BIIB033 is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for LINGO binding with BIIB033 are also encompassed by the invention.

In a specific embodiment, an MRD-containing antibody binds LINGO and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3, TNFRSF21 (DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19, CD22, TNFRSF5 (CD40), CD80, cMET, NRP1, TNF, TNFSF12 (TWEAK), HGF, IGF1, IGF1,2, IGF2, NGF, Te38, NogoA, RGM A, MAG, OMGp, NgR, NGF, PGE2, IL1 beta, Semaphorin 3A and Semaphorin 4. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind LINGO and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds LINGO. In further embodiments, the antibody component of the MRD-containing antibody is BIIB033.

In a specific embodiment, the target of an antibody of an MRD-containing antibody is TNFSF12 (TWEAK). In another embodiment, the antibody in the MRD-containing antibody binds TNFSF12 (TWEAK) and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3, TNFRSF21 (DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19, CD22, TNFRSF5 (CD40), CD80, cMET, NRP1, TNF, LINGO, HGF, IGF1, IGF1,2, IGF2, NGF, Te38, NogoA, RGM A, MAG, OMGp, NgR, NGF, PGE2, IL1 beta, Semaphorin 3A and Semaphorin 4. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNFSF12 (TWEAK) and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNFSF12 (TWEAK). In further embodiments, the antibody component of the MRD-containing antibody is BIIB023.

In another embodiment, the target of the antibody of the MRD-containing antibody is: oxidized LDL, gpIIB, gpIIIa, PCSK9, Factor VIII, integrin a2bB3, AOC3, or mesothelin. In specific embodiments, the antibody in the MRD-containing antibody is BI-204 oxidized LDL (BioInvent), abciximab gpIIB, gpIIIa (e.g., REOPRO, Eli Lilly), AMG-145 PCSK9 (Amgen), TB-402 Factor VIII (BioInvent), vapaliximab, or tadocizumab integrin a2bB3 (Yamonochi Pharma). In another embodiment, the antibody specifically binds to the same epitope as BI-204, abciximab, AMG-145, TB-402, or tadocizumab. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits binding of BI-204, abciximab, AMG-145, TB-402, vapaliximab, or tadocizumab. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In other embodiments, the antibody of the MRD-containing antibody is associated with bone growth and/or metabolism. In certain embodiments the antibody target of the MRD-containing antibody is TNFSF11 (RANKL). In other embodiments the antibody target of the MRD-containing antibody is: DKK1, osteopontin, cathepsin K, TNFRSF19L (RELT), TNFRSF19 (TROY), or sclerostin (CDP-7851 UCB Celltech). In another embodiment antibody target of the MRD-containing antibody is TNFSF11 (RANKL). In a specific embodiment, the antibody in the MRD-containing antibody is denosumab (e.g., AMG-162, Amgen). In another embodiment, the antibody specifically binds to the same epitope as denosumab. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits binding of TNFSF11 (RANKL) by denosumab. In another specific embodiment, the antibody is AMG617 or AMG785 (e.g., CDP7851, Amgen). In another embodiment, the antibody specifically binds to the same epitope as AMG617 or AMG785. In another embodiment, the antibody in the MRD-containing antibody is an antibody that competitively inhibits binding of sclerostin by AMG617 or AMG785. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In one embodiment, an MRD-containing antibody binds TNFSF11 (RANKL). In a specific embodiment, an MRD-containing antibody binds TNFSF11 and a target selected from: sclerostin (SOST), endothelin-1, DKK1, IL1, IL6, IL7, IL8, IL11, IL17A, MCSF, IGF1, IGF2, IGF1,2 IGF1R, TNF, FGF1, FGF2, FGF4, FGF7, FGF8a, FGF8b, FGF18, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TGF beta, TGF beta R2, BMP2, BMP4, BMP5, BMP9, BMP10, BMPR-IA, PDGF, PDGFRa, PDGFRb PTH, PTH related protein (PTHrP), and PGE2. In a particular embodiment, the MRD-containing antibody binds TNFSF11 and DKK1. In a further embodiment the antibody of the MRD-containing antibody binds DKK1. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNFSF11 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNFSF11. In further embodiments, the antibody component of the MRD-containing antibody is denosumab, AMG617 or AMG785.

In additional embodiments, the antibody target of the MRD-containing antibody is a bacterial antigen, a viral antigen, a mycoplasm antigen, a prion antigen, or a parasite antigen (e.g., one infecting a mammal).

In other embodiments, the target of the antibody of the MRD-containing antibody is a viral antigen. In one embodiment, the target of the antibody of the MRD-containing antibody is anthrax, hepatitis b, rabies, Nipah virus, west nile virus, a mengititis virus, or CMV. In other embodiments, the antibody of the MRD-containing antibody competes with antigen binding with ABTHRAX® (Human Genome Sciences), exbivirumab, foravirumab, libivirumab, rafivirumab, regavirumab, sevirumab (e.g., MSL-109, Protovir), tuvirumab, raxibacumab, Nipah virus M102.4, or MGAWN1® (MacroGenics) for target binding. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In other embodiments, the target of the antibody of the MRD-containing antibody is RSV. In other embodiments, the antibody of the MRD-containing antibody is motavizumab (e.g., NUMAX®, MEDI-577; MedImmune) or palivizumab RSV fusion f protein (e.g., SYNAGIS®, MedImmune). In other embodiments, the antibody of the MRD-containing antibody competes with motavizumab or palivizumab RSV fusion f protein, for target binding. In other embodiments, the antibody of the MRD-containing antibody is felvizumab. In other embodiments, the antibody of the MRD-containing antibody competes with felvizumab for target binding. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In other embodiments, the target of the antibody of the MRD-containing antibody is a bacterial or fungal antigen. In other embodiments, the antibody of the MRD-containing antibody competes for antigen binding with nebacumab, edobacomab (e.g., E5), tefibazumab (Inhibitex), panobacumab (e.g., KBPA101, Kenta), pagibaximab (e.g., BSYX-A110, Biosynexus), urtoxazumab, or efungumab (e.g., MYCOGRAB®, Novartis). In other embodiments, the antibody of the MRD-containing antibody is nebacumab, edobacomab, tefibazumab (Inhibitex), panobacumab, pagibaximab, urtoxazumab, or efungumab. An MRD that competes for target binding with one of the above antibodies is also encompassed by the invention. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with 1, 2 or more of the above antibodies are also encompassed by the invention.

In another specific embodiment, the antibody in the MRD-containing antibody is the catalytic antibody 38C2. In another embodiment, the antibody binds to the same epitope as 38C2. In another embodiment, the antibody competitively inhibits 38C2.

Other antibodies of interest include A33 binding antibodies. Human A33 antigen is a transmembrane glycoprotein of the Ig superfamily. The function of the human A33 antigen in normal and malignant colon tissue is not yet known. However, several properties of the A33 antigen suggest that it is a promising target for immunotherapy of colon cancer. These properties include (i) the highly restricted expression pattern of the A33 antigen, (ii) the expression of large amounts of the A33 antigen on colon cancer cells, (iii) the absence of secreted or shed A33 antigen, (iv) the fact that upon binding of antibody A33 to the A33 antigen, antibody A33 is internalized and sequestered in vesicles, and (v) the targeting of antibody A33 to A33 antigen expressing colon cancer in preliminary clinical studies. Fusion of a MRD directed toward A33 to a catalytic or non-catalytic antibody would increase the therapeutic efficacy of A33 targeting antibodies.

In some embodiments, the antibody in the MRD-containing antibody binds to a human target protein. In some embodiments, the MRD binds to both a human protein and its ortholog in mouse, rat, rabbit, or hamster.

The antibodies in the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are able to bind their respective targets when the MRDs are attached to the antibody. In certain embodiments, the antibody binds its target independently. In some embodiments, the antibody is a target agonist. In other embodiments, the antibody is a target antagonist. In certain embodiments, the antibody can be used to localize an MRD-containing antibody to an area where the antibody target is located.

It is contemplated that the antibodies used in the present invention may be prepared by any method known in the art. For example, antibody molecules and multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be “recombinantly produced,” i.e., produced using recombinant DNA technology.

Monoclonal antibodies that can be used as the antibody component of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in vitro, for example, using known methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo, for example, as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinant DNA methods, for example, as described in U.S. Pat. No. 4,816,567. For example, in one approach polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. In other approaches, recombinant monoclonal antibodies or antibody fragments having the desired immunoreactivity can be isolated from phage display libraries expressing CDRs of the desired species using techniques known in the art (McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)).

The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different ways, using recombinant DNA technology to generate alternative antibodies. For example, polynucleotide sequences that encode one or more MRDs and optionally linkers, can be operably fused, for example, to the 5′ or 3′ end of sequence encoding monoclonal antibody sequences. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted (1) for those regions of for example, a human antibody to generate a chimeric antibody or (2) for a non-immunoglobulin polypeptide to generate a fusion antibody. Techniques for site-directed and high-density mutagenesis of the variable region are known in the art and can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

In certain embodiments, the antibody of the MRD-containing antibody is a human antibody. For example, human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (1):86-95 (1991); and U.S. Pat. Nos. 5,750,373 and 6,787,637). In one embodiment, the human antibody can be derived from the “minilocus approach” in which an exogenous Ig locus is mimicked through inclusion of individual genes from the Ig locus (see e.g., U.S. Pat. No. 5,545,807). Methods of preparing a human antibody from a phage library, and optionally optimizing binding affinity are known in the art and described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998); Hoogenboom et al., Nat. Biotechnology 23:1105-1116 (2005); Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Persic et al., Gene 187:9-18 (1997); Jostock et al., J. Immunol. Methods 289:65-80 (2004); Marks et al., J. Mol. Biol., 222:581 (1991)); et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); et al., Proc. Natl. Acad. Sci. USA 91:3809-3813 (1994); Yang et al., J. Mol. Biol. 254:392-403 (1995); and Barbas et al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992). Techniques for the generation and use of antibody phage libraries are also described in: U.S. Pat. Nos. 5,545,807, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, and 7,264,963; and Rothe et al., J. Mol. Bio. 130:448-54 (2007) (each of which is herein incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., Bio/Technology 10:779-783 (1992) (which is herein incorporated by reference in its entirety) are known in the art and can be employed to generate high affinity human antibodies.

Antibodies can also be made in mice that are transgenic for human immunoglobulin genes or fragments of these genes and that are capable, upon immunization, of producing a broad repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in: Lonberg, Nat. Biotechnol 23:1117-1125 (2005), Green et al., Nature Genet. 7:13-21 (1994), and Lonberg et al., Nature 368:856-859 (1994); U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 6,596,541, 7,105,348, and 7,368,334 (each of which is herein incorporated by reference in its entirety).

IV. Linkers

Multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention can contain a single linker, multiple linkers, or no linker. Thus, a MRD may be operably attached (linked) to the antibody directly, or operably attached through an optional linker peptide. Similarly, a MRD may be operably attached to one or more MRD(s) directly, or operably attached to one or more MRD(s) through one or more optional linker peptide(s). Linkers can be of any size or composition so long as they are able to operably attach an MRD and an antibody such that the MRD enables the MRD containing antibody to bind the MRD target.

In some embodiments, linkers have about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The linker can also have about 4 to 15 amino acids. In certain embodiments, the linker peptide contains a short linker peptide with the sequence GGGS (SEQ ID NO:1), a medium linker peptide with the sequence SSGGGGSGGGGGGSS (SEQ ID NO:2), or a long linker peptide with the sequence SSGGGG SGGGGGGSSRSS (SEQ ID NO:19). In another embodiment, the MRD is inserted into the fourth loop in the light chain constant region. For example, the MRD can be inserted between the underlined letters in the following amino acid sequence: RTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDKLGTNSQESVTEQDSKDSTYSL SS TLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC (SEQ ID NO:102).

The linker can also be a non-peptide linker such as an alkyl linker, or a PEG linker. For example, alkyl linkers such as —NH—(CH₂)s-C(O)—, wherein s=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In certain embodiments, the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa.

In some embodiments, the linker is a “cleavable linker” facilitating release of an MRD or cytotoxic agent in the cell. For example, an acid-labile linker (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020; U.S. Appl. Pub. No. 20090110753) can be used wherein it is desirable that the covalent attachment between an MRD or a cytoxic agent and the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is intracellularly cleaved when the composition is internalized into the cell. The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on an antibody-drug conjugate (ADC) whereby the covalent attachment, i.e., linked via a linker between the MRD and cytotoxic agent, MRD and antibody, antibody and cytotoxic agent, or between two MRDs is broken, resulting in the free MRD and/or cytotoxic agent dissociated from the antibody inside the cell. The cleaved moieties of the zybody-ADC are thus intracellular metabolites.

Linker optimization can be evaluated using the techniques described in Examples 1-18 and techniques otherwise known in the art. Linkers preferably should not disrupt the ability of an MRD and/or an antibody to bind target molecules.

V. Antibodies Containing MRDs

Using the methods described herein, multi-specificity and greater multi-valency can be achieved through the fusion of MRDs to antibodies.

The MRDs of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) prepared according to the present invention, may be operably linked to an antibody through the peptide's N-terminus or C-terminus. The MRD may be operably linked to the antibody at the C-terminal end of the heavy chain of the antibody, the N-terminal end of the heavy chain of the antibody, the C-terminal end of the light chain of the antibody, or the N-terminal end of the light chain of the antibody. Optimization of the MRD composition, MRD-antibody attachment location and linker composition can be performed using the binding assays described in Examples 1-18 and bioassays and other assays known in the art for the appropriate target related biological activity.

In one embodiment, an MRD-containing antibody is an MRD-containing antibody described in U.S. Application No. 61/489,249, filed May 24, 2011, which is herein incorporated by reference in its entirety.

In one embodiment, multivalent and multispecific compositions (e.g., MRD-containing antibodies) contain an MRD operably linked to either the antibody heavy chain, the antibody light chain, or both the heavy and the light chain. In one embodiment, an MRD-containing antibody contains at least one MRD linked to one of the antibody chain terminals. In another embodiment, an MRD-containing antibody of the invention contains at least one MRD operably linked to two of the antibody chain terminals. In another embodiment, an MRD-containing antibody contains at least one MRD operably linked to three of the antibody chain terminals. In another embodiment, an MRD-containing antibody contains at least one MRD operably attached to each of the four antibody chain terminals (i.e., the N and C terminals of the light chain and the N and C terminals of the heavy chain).

In certain specific embodiments, the MRD-containing antibody has at least one MRD operably attached to the N-terminus of the light chain. In another specific embodiment, the MRD-containing antibody has at least one MRD operably attached to the N-terminus of the heavy chain. In another specific embodiment, the MRD-containing antibody has at least one MRD operably attached to the C-terminus of the light chain. In another specific embodiment, the MRD-containing antibody has at least one MRD operably attached to the C-terminus of the heavy chain.

An MRD-containing antibody can be “multispecific” (e.g., bispecific, trispecific tetraspecific, pentaspecific or of greater multispecificity), meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins). Thus, whether an MRD-containing antibody is “monospecific” or “multispecific,” (e.g., bispecific, trispecific, and tetraspecific) refers to the number of different epitopes that the MRD-containing antibody binds. Multispecific antibodies may be specific for different epitopes of a target polypeptide (e.g., as described herein) or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide target or solid support material. The present invention contemplates the preparation of mono-, bi-, tri-, tetra-, and penta-specific antibodies as well as antibodies of greater multispecificity. In one embodiment, the MRD-containing antibody binds two different epitopes. In an additional embodiment the MRD-containing antibody binds two different epitopes simultaneously. In another embodiment, the MRD-containing antibody binds three different epitopes. In an additional embodiment the MRD-containing antibody binds three different epitopes simultaneously. In another embodiment, the MRD-containing antibody binds four different epitopes. In an additional embodiment the MRD-containing antibody binds four different epitopes simultaneously. In another embodiment, the MRD-containing antibody binds five different epitopes (see, e.g., FIG. 2D). In an additional embodiment the MRD-containing antibody binds five different epitopes simultaneously.

In other embodiments two MRDs of the MRD-containing antibody bind the same antigen. In other embodiments three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same antigen. In other embodiments at least two MRDs of the MRD-containing antibody bind the same antigen. In other embodiments at least three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same antigen. In other embodiments two MRDs of the MRD-containing antibody bind the same epitope. In other embodiments three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same epitope. In other embodiments at least two MRDs of the MRD-containing antibody bind the same epitope. In other embodiments at least three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same epitope.

In other embodiments, the antibody and one MRD of the MRD-containing antibody bind the same antigen. In other embodiments the antibody and two, three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same antigen. In other embodiments, the antibody and at least one MRD of the MRD-containing antibody bind the same antigen. In other embodiments the antibody and at least two, three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same antigen. In other embodiments, the antibody and one MRD of the MRD-containing antibody bind the same epitope. In other embodiments the antibody and two, three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same epitope. In other embodiments, the antibody and at least one MRD of the MRD-containing antibody bind the same epitope. In other embodiments the antibody and at least two, three, four, five, six, seven, eight, nine or ten MRDs of the MRD-containing antibody bind the same epitope.

The present invention also provides for two or more MRDs which are linked to any terminal end of the antibody. Thus, in one non-exclusive embodiment, two, three, four, or more MRDs are operably linked to the N-terminal of the heavy chain. In another non-exclusive embodiment, two, three, four, or more MRDs are operably linked to the N-terminal of the light chain. In another non-exclusive embodiment, two, three, four, or more MRDs are operably linked to the C-terminal of the heavy chain. In another non-exclusive embodiment, two, three, four, or more MRDs are operably linked to the C-terminal of the light chain. It is envisioned that these MRDs can be the same or different. In addition, any combination of MRD number and linkages can be used. For example, two MRDs can be operably linked to the N-terminal of the heavy chain of an antibody which contains one MRD linked to the C-terminal of the light chain. Similarly, three MRDs can be operably linked to the C-terminal of the light chain and two MRDs can be operably linked to the N-terminal of the light chain.

Multivalent and multispecific compositions (e.g., MRD-containing antibodies) can contain one, two, three, four, five, six, seven, eight, nine, ten or more than ten MRDs.

In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains one MRD (see, e.g., FIGS. 2B and 2C). In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains two MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains three MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains four MRDs (see, e.g., FIGS. 2B and 2C). In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains five MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains six MRDs. In an additional embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains between two and ten MRDs.

In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one MRD. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least two MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least three MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least four MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least five MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least six MRDs.

In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains two different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains three different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains four different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains five different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains six different MRDs. In an additional embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains between two and ten different MRDs.

In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least two different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least three different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least four different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least five different MRDs. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least six different MRDs.

Thus, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be MRD monomeric (i.e., containing one MRD at the terminus of a peptide chain optionally connected by a linker) or MRD multimeric (i.e., containing more than one MRD in tandem optionally connected by a linker). The multimeric multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be homo-multimeric (i.e., containing more than one of the same MRD in tandem optionally connected by linker(s) (e.g., homodimers, homotrimers, homotetramers etc.)) or hetero-multimeric (i.e., containing two or more MRDs in which there are at least two different MRDs optionally connected by linker(s) where all or some of the MRDs linked to a particular terminus are different (e.g., heterodimer, heterotrimer, heterotetramer etc.)). In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains two different monomeric MRDs located at different immunoglobulin termini. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains three different monomeric MRDs located at different immunoglobulin termini. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains four different monomeric MRDs located at different immunoglobulin termini. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains five different monomeric MRDs located at different immunoglobulin termini. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains six different monomeric MRDs located at different immunoglobulin termini.

In an alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one dimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one homodimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one heterodimeric and one monomeric MRD located at different immunoglobulin termini.

In an alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one multimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one homomultimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains at least one heteromultimeric and one monomeric MRD located at different immunoglobulin termini.

In an alternative embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) contains MRDs operably linked to at least two different immunoglobulin termini. In a specific embodiment, the MRDs fused to at least one of the immunoglobulins are a multimer. In one embodiment, the MRDs fused to a least one of the immunoglobulins are a homomultimeric (i.e., more than one of the same MRD operably linked in tandem, optionally linked via a linker). In another embodiment, the MRDs fused to at least one of the immunoglobulins are a heteromultimeric (i.e., two or more different MRDs operably linked in tandem, optionally linked via a linker). In an additional embodiment, the MRDs fused to at least one of the immunoglobulins are a dimer. In another embodiment, the MRDs fused to a least one of the immunoglobulins are a homodimer. In another embodiment, the MRDs fused to at least one of the immunoglobulins are a heterodimer.

The multiple MRDs can target the same target binding site, or two or more different target binding sites. Where the MRDs bind to different target binding sites, the binding sites may be on the same or different target molecules.

Similarly, the antibody and the MRD in a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) may bind to the same target molecule or to different target molecules.

In some embodiments, at least one MRD and the antibody in the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) can bind to their targets simultaneously. In one embodiment, each MRD in the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) and the antibody can bind to its target simultaneously. Therefore, in some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds two, three, four, five, six, seven, eight, nine, ten or more targets simultaneously.

The ability of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to bind to multiple targets simultaneously can be assayed using methods known in the art, including, for example, those methods described in the examples below.

Multivalent and Multispecific Compositions Having Monovalent Specificity

In additional embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention have a single binding site for (i.e., monovalently bind) a target.

In some embodiments, the antigen binding domains of an antibody component of a multivalent and monovalent multispecific composition of the invention binds to different target epitopes (i.e., the antibody is bispecific). The term “bispecific antibody” is intended to include any antibody, which has two different binding specificities, i.e. the antibody binds two different epitopes, which may be located on the same target antigen or, more commonly, on different target antigens. Methods for making bispecific antibodies are known in the art. (See, for example, Millstein et al., Nature, 305:537-539 (1983); Traunecker et al., EMBO J. 10:3655-3659 (1991); Suresh et al., Methods in Enzymology 121:210 (1986); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893, 4,676,980, 4,714,681, 4,925,648, 5,573,920, 5,601,819, 5,731,168, 5,807,706, and 5,821,333; Intl. Appl. Publ. Nos. WO94/04690, WO91/00360, WO92/05793, WO92/08802, WO92/200373, WO93/17715, WO00/44788, and WO02/096948; EP 1870459A1 and EP 03089, the contents of each of which is herein incorporated by reference in its entirety).

One method for generating bispecific antibodies has been termed the “knobs-into-holes” strategy (see, e.g., Intl. Publ. WO2006/028936). The mispairing of Ig heavy chains is reduced in this technology by mutating selected amino acids forming the interface of the CH3 domains in IgG. At positions within the CH3 domain at which the two heavy chains interact directly, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain and an amino acid with a large side chain (knob) into the counterpart interacting residue location on the other heavy chain. In some embodiments, compositions of the invention have immunoglobulin chains in which the CH3 domains have been modified by mutating selected amino acids that interact at the interface between two polypeptides so as to preferentially form a bispecific antibody. The bispecific antibodies can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4)

In one embodiment, a bispecific antibody component of a multispecific and multivalent composition (e.g., MRD-containing antibody) comprises a T366W mutation in the “knobs chain” and T366S, L368A, Y407V mutations in the “hole chain,” and optionally an additional interchain disulfide bridge between the CH3 domains by, e.g., introducing a Y349C mutation into the “knobs chain” and a E356C mutation or a S354C mutation into the “hole chain;” R409D, K370E mutations in the “knobs chain” and D399K, E357K mutations in the “hole chain;” R409D, K370E mutations in the “knobs chain” and D399K, E357K mutations in the “hole chain;” a T366W mutation in the “knobs chain” and T366S, L368A, Y407V mutations in the “hole chain;” R409D, K370E mutations in the “knobs chain” and D399K, E357K mutations in the “hole chain;” Y349C, T366W mutations in one of the chains and E356C, T366S, L368A, Y407V mutations in the counterpart chain; Y349C, T366W mutations in one chain and S354C, T366S, L368A, Y407V mutations in the counterpart chain; Y349C, T366W mutations in one chain and S354C, T366S, L368A, Y407V mutations in the counterpart chain; and Y349C, T366W mutations in one chain and S354C, T366S, L368A, Y407V mutations in the counterpart chain (numbering according to the EU index of Kabat).

In some embodiments, a bispecific antibody component of a composition of the invention (e.g., MRD-containing antibody) is an IgG4 antibody or a modified IgG4 antibody, or contains an IgG4 heavy chain or a modified IgG4 heavy chain. IgG4 antibodies are dynamic molecules that undergo Fab arm exchange by swapping an IgG4 heavy chain and attached light chain for a heavy-light chain pair from another IgG4 molecule, thus resulting in bispecific antibodies. Accordingly, Fab arm exchange by swapping of MRD-containing-IgG4 antibodies whether caused in vivo or in vitro under physiologic conditions will lead to bispecific antibody compositions. In particular embodiments, an IgG4 heavy chain of a composition of the invention contains an S228P substitution. This substitution has been shown to significantly inhibit Fab arm exchange in the resulting mutant IgG4 antibodies, and to thereby reduce the likelihood of Fab-arm-exchange between a recombinant antibodies and endogenous IgG4. (See, e.g., Labrijn et al., Nat. Biotechnol. 27(8):767-71 (2009)). In additional embodiments, an IgG4 heavy chain of a composition of the invention contains a substitution of the Arg at position 409 (e.g., with Lys, Ala, Thr, Met or Leu), the Phe at position 405 (e.g., with Lys, Ala, Thr, Met or Leu) or the Lys at position 370. In other embodiments, the CH3 region of an IgG4 heavy chain of a composition of the invention has been replaced with the CHH3 region of IgG1, IgG2 or IgG3. In additional embodiments, interactions between one or more MRDs located at the C-termini of distinct heavy chains (e.g., IgG4 or IgG4 and IgG3) favor and/or stabilize heterodimers between the heavy chains, or otherwise reduces Fab arm exchange by the heterodimer.

Exemplary bispecific antibody components of multivalent and multispecific compositions of the invention include, IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG2, IgG4 and IgG3, IgG1 and IgG3 chain heterodimers. Such heterodimeric heavy chain antibodies, can routinely be engineered by, for example, modifying selected amino acids forming the interface of the CH3 domains in human IgG4 and the IgG1 or IgG3 so as to favor heterodimeric heavy chain formation. In additional embodiments, interactions between one or more MRDs located at the C-termini of heteromeric heavy chains favors or stabilizes heteromultimeric formation or structure, respectively.

IgG4 antibodies are known to have decreased ADCC activity and half-life compared to other immunoglobulins subclasses such as, IgG1 and IgG3. Accordingly, IgG4 subclass-based formats provide an attractive format for developing therapeutics that bind to and block cell receptors, but do not deplete the target cell. Alternatively, in those embodiments for which increased effector activity is desired, an IgG4 heavy chain of a composition of the invention can be modified as described herein or otherwise known in the art, so as to increase effector function (e.g., modification of the residues at positions 327, 330 and 331; numbering according to EU index of Kabat). Similarly, where increased half-life is desired, an IgG4 heavy chain of a composition of the invention can be engineered as described herein, or otherwise known in the art to more selectively bind the FcRn at pH 6.0, but not pH 7.4, by for example, incorporating mutations located at the interface between the CH2 and CH3 domains, such as substitutions at T250Q/M428L as well as M252Y/S254T/T256E and H433K/N434F (numbering according to the EU index of Kabat).

As exemplified above, it is envisioned that in some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention have a single binding site for (i.e., monovalently bind) a target. In some embodiments, the single binding site (i.e., monovalent binding site) is an antibody antigen binding domain. In other embodiments, the single binding site is an MRD. Thus, the multivalent and multispecific compositions of the invention encompass (and can be routinely engineered to include) MRD-containing antibodies that that contain 1, 2, 3, 4 or more single binding sites for a target. The single binding site(s) may be provided by one or more MRDs located at any one or more of the 4 immunoglobulin heavy chain termini or 4 immunoglobulin light chain termini. Moreover, single binding site may be provided by one of the antigen binding domains of the antibody (wherein an MRD of the MRD-containing antibody binds the same target epitope of the other antigen binding domain of the antibody. Moreover, in a specific embodiment, the compositions of the invention encompass (and can be routinely engineered to include) MRD-containing antibodies that contain 1, 2, 3, 4 or more single binding sites for a target and do not bivalently bind another target

In further embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) has a single binding site for (i.e., monovalently binds) a cell surface target that forms multimers (e.g., homomers or heteromers). In some embodiments, the single binding site binds a cell surface target that requires multimerization for signaling. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has a single binding site that binds a cell surface target and inhibits binding of another molecule (such as a ligand) to the cell surface target. In other embodiments, binding of the single binding site inhibits multimerization of the target (e.g., homomeric and heteromeric multimerization). In additional embodiments, the composition has single binding sites for different targets (i.e., monovalently binds more than one different target). In some embodiments, the multiple single binding sites of the composition bind targets on the same cell. In additional embodiments, the multiple single binding sites of the composition bind targets on different cells. Numerous receptors are known in the art that require multimerization for affecting their normal function. Such receptors are envisioned to be targets of single binding sites in the multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention. In some embodiments, the composition has a single binding site for a receptor tyrosine kinase. In some embodiments, the composition has a single binding site for a growth factor receptor. In additional embodiments the composition has a single binding site for a G protein coupled receptor. In additional embodiments the composition has a single binding site for a chemokine receptor. In other embodiments, the composition has a single binding site for a TNF receptor superfamily member. In particular embodiments, the composition has a single binding site for a receptor selected from: RAGE, c-Met, ErbB2, VEGFR1, VEGFR2, VEGFR3, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, PDGFRA, PDGFRB, netrin, CD28, TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF21 or TNFRSF25, TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFRSF21 (DR6), TNFRSF25 (DR3), and LRP6.

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has a single binding site for (i.e., monovalently binds) a cell surface target that forms a multimer and multiple sites (i.e., multivalently binds) for two or more different targets. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for a cell surface target and multiple binding sites for 1, 2, 3, 4, 5 or more different targets. In further embodiments, at least 1, 2, 3, 4, 5 or more of the targets bound by the multivalent and monovalent multispecific composition are located on a cell surface. In other embodiments, at least 1, 2, 3, 4, 5 or more of the targets bound by the multivalent and monovalent multispecific composition are soluble targets (e.g., chemokines, cytokines, and growth factors). In additional embodiments, the composition binds 1, 2, 3, 4, 5 or more of the targets described herein. In further embodiments, the targets bound by the composition are tumor antigens (including tumor antigens and tumor associated antigens). In additional embodiments, a target bound by the composition is associated with a disease or disorder of the immune system. In further embodiments, a targets bound by the composition is associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In some embodiments, an MRD-containing antibody has a single binding site for TNFRSF21 (DR6). In further embodiments, the MRD-containing antibody has a single binding site for DR6 and binds a target selected from: AGE (S100 A, amphoterin), IL1, IL6, IL18, IL12, IL23, TNFSF12 (TWEAK), TNF alpha, VEGF, TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), interferon gamma, GMCSF, an FGF, CXCL13, MCP 1, CCR2, NogoA, RGM A, OMgp MAG, a CPSG, LINGO, alpha-synuclein, a semaphorin (e.g., Semaphorin 3A, Semaphorin 4), an ephrin, VLA4, CD45, RB, C5, CD52 and CD200. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind DR6 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. These compositions have applications in treating diseases and disorders including neurological diseases and disorders such as multiple sclerosis and other neurodegenerative diseases. In specific embodiments, the antibody component of the MRD-containing antibody binds DR6.

In some embodiments, an MRD-containing antibody has a single binding site for TNFRSF25 (DR3). In further embodiments, the MRD-containing antibody has a single binding site for DR3 and binds a target selected from: TNF, IFN alpha, IFN gamma, IL1, IL1beta, IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind DR3 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. These compositions have applications in treating diseases and disorders including inflammatory bowel disease and autoimmune diseases such as rheumatoid arthritis. In specific embodiments, the antibody component of the MRD-containing antibody binds DR3.

In further embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) has multiple binding site for (i.e., multivalently binds) a cell surface target that forms multimers (e.g., homomers or heteromers). In some embodiments, the multiple binding sites bind a cell surface target that requires multimerization for signaling. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has multiple binding sites for a cell surface target. In further embodiments, binding of the multiple binding sites result in multimerization of the target (e.g., homomeric and heteromeric multimerization). In additional embodiments, the composition has multiple binding sites for different targets (i.e., multivalently binds more than one different target). In some embodiments, the multiple single binding sites of the composition bind targets on the same cell. In additional embodiments, the multiple single binding sites of the composition bind targets on different cells. Numerous receptors are known in the art that require multimerization for affecting their normal function. Such receptors are envisioned to be targets of the multivalent and multispecific compositions (e.g., MRD-containing antibodies). In some embodiments, the composition has multiple binding sites for a receptor tyrosine kinase. In some embodiments, the composition has a multiple binding site for a growth factor receptor. In additional embodiments the composition has multiple binding sites for a G protein coupled receptor. In additional embodiments the composition has multiple binding sites for a chemokine receptor. In other embodiments, the composition has multiple binding sites for a TNF receptor superfamily member.

In some embodiments, an MRD-containing antibody binds TNFRSF10A (DR4). In further embodiments, the MRD-containing antibody binds DR4 and a target selected from: ErbB2, EGFR, IGF1R, TNFRSF10b (DR5), CD19, CD20, CD22, CD30, CD33, TNFRSF5 (CD40), TNFRSF9 (41BB), IL6, and IGF1,2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind DR4 and also bind at least 2, 3, 4, 5 or more of these targets are also encompassed by the invention. These compositions have applications in treating diseases and disorders including cancers such as breast cancer, colorectal cancer, head and neck cancer, B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia and melanoma. In specific embodiments, the antibody component of the MRD-containing antibody binds DR4. In further embodiments, the antibody component of the MRD-containing antibody is CS1008 or mapatumumab.

In some embodiments, an MRD-containing antibody binds TNFRSF10B (DR5). In some embodiments, an MRD-containing antibody binds DR5 and a target selected from: ErbB2, EGFR, IGF1R, TNFRSF10A (DR4), CD19, CD20, CD22, CD25, CD30, CD33, CD138, syndecan, CD39, TNFRSF5 (CD40), TNFRSF9 (41BB), IL6, and IGF1,2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind DR5 and also bind at least 2, 3, 4, 5 or more of these targets are also encompassed by the invention. These compositions have applications in treating diseases and disorders including cancers such as breast cancer, colorectal cancer, head and neck cancer, B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma. In specific embodiments, the antibody component of the MRD-containing antibody binds DR5. In further embodiments, the antibody component of the MRD-containing antibody is LBY135, AMG66, Apomab, PRO95780, lexatumumab, conatumumab or tigatuzumab.

Compositions that Redirect Effector Cell Function

The invention also encompasses multivalent and multispecific compositions such as, multivalent and multispecific compositions (e.g., MRD-containing antibodies) that are capable of juxtaposing host effector cells with cells that are desired to be eliminated (e.g., immune cells, cancer cells, diseased cells, infectious agents, and cells infected with infectious agents). The multivalent and multispecific functionalities of the compositions of the invention are particularly well suited for redirecting host immune responses and provide numerous advantages over alternative multispecific composition platforms under development. In one embodiment, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., an immune cell or a tumor antigen on a tumor cell) and (2) a target on an effector cell so as to direct an immune response to the cell, tissue, or infectious agent of interest. The target(s) to which the multivalent and monovalent multispecific composition binds can be monomeric or multimeric. Moreover, the mulitimeric target to which a multivalent and monovalent multispecific composition binds can be homomultimeric or heteromultimeric. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. In additional embodiments, one or more targets bound by the multivalent and monovalent multispecific composition is a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). The multivalent and multispecific compositions also have applications in treating diseases and disorders including, but not limited to, diseases of the immune system, skeletal system, cardiovascular system, and nervous system, as well as infectious disease. Thus, in some embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is associated with a disease or disorder of the immune system (for example, a disease or disorder of the immune system disclosed herein, such as inflammation or an autoimmune disease (e.g., rheumatoid arthritis)). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is associated with a disease or disorder of the skeletal system (e.g., osteoporosis or another disease or disorder of the skeletal system as disclosed herein). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is associated with a disease or disorder of the cardiovascular system (e.g., a disease or disorder of the cardiovascular system disclosed herein). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is associated with a disease or disorder of the nervous system (e.g., a disease or disorder of the nervous system disclosed herein). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is associated with an infectious agent or disease (e.g., an infectious disease or agent disclosed herein).

Effector cells that can be bound by a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) of the invention include, but are not limited to, T cells, monocytes/macrophages, and natural killer cells.

In one embodiment, the target on a cell to which a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) directs an immune response is a tumor antigen. The multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies) are envisioned to be capable of binding virtually any type of tumor and any type of tumor antigen. Exemplary types of tumors that can be targeted include, but are not limited to, one or more cancers selected from the group: colorectal cancer, esophageal, gastric, head and neck cancer, thyroid cancer, multiple myeloma, renal cancer, pancreatic cancer, lung cancer, biliary cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer breast cancer, ovarian cancer, cervical cancer, and endometrial cancer. Exemplary types of tumors that may be targeted include hematological cancers. Hematological cancers that may be targeted include, but are not limited to, one or more cancers selected from the group Hodgkin's lymphoma, medullary non-Hodgkin's lymphoma, acute lymphoblastic leukemia, lymphocytic leukemia, and chronic myelogenous leukemia, acute myelogenous leukemia.

Exemplary tumor antigens include ErbB1, ErbB2, ErbB3, VEGFR1, VEGFR2, EGFRvIII, CD16, CD19, CD20, oncostatin M, PSA, PSMA, integrin avb6, ADAM9, CD22, CD23, CD25, CD28, CD36, CD45, CD46, CD56, CD79a/CD79b, CD103, JAM-3, gp100, ALCAM, PIPA, A33, carboxypeptidease M, E-cadherin, CA125, CDK4, CEA, CTLA-4, RAAG10, transferrin receptor, p-15, GD2, MUM-1, MAGE-1, MAGE-3, KSA, MOC31, MIC-1, EphA2, GAGE-1, GAGE-2, MART, KID31, CD44v3, CD44v6, and ROR1. Additional exemplary tumor antigens are described herein and/or known in the art.

In one embodiment, the target on a cell to which a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) directs an immune response is an immune cell or an inflammatory cell.

In some embodiments, the invention encompasses a multivalent and monovalent multispecific composition that binds a tumor antigen that is not expressed on tumor cells themselves, but rather on the surrounding reactive and tumor supporting, non-malignant cells comprising the tumor stroma (i.e., tumor associated antigens). The tumor stroma comprises endothelial cells forming new blood vessels and stromal fibroblasts surrounding the tumor vasculature. In one embodiment, a multivalent and monovalent multispecific composition binds a tumor associated antigen on an endothelial cell. In an additional embodiment, a multivalent and monovalent multispecific composition binds a tumor antigen and also binds a tumor associated antigen on a fibroblast cell. In a further embodiment, a multivalent and monovalent multispecific composition binds a tumor antigen and also binds fibroblast activation protein (FAP).

Infectious agents to which a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) can direct an immune response include, but are not limited to, prokaryotic and eukaryotic cells, viruses (including bacteriophage), foreign objects (e.g., toxins), and infectious organisms such as funghi, and parasites (e.g., mammalian parasites), as described herein and infectious agents associated with infectious diseases described herein. The term infectious agents is also intended to encompass other prokaryotic and eukaryotic cells, viruses (including bacteriophage), foreign objects (e.g., toxins), and infectious organisms such as funghi, and parasites otherwise known in the art.

In further embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) has a single binding site for a target on an effector cell so as to direct an immune response to the cell, tissue, or infectious agent of interest. In some embodiments the single binding site is an MRD. In other embodiments, the single binding site is an antibody antigen binding domain. In further embodiments, binding of the multivalent and monovalent multispecific composition does not elicit a signal when the composition binds a target on an effector cell. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). In additional embodiments, one or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, one or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) a target on a leukocyte so as to direct an immune response to the cell, tissue, or infectious agent of interest. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments the multivalent and monovalent multispecific composition binds 1, 2, 3, 4, 5 or more targets described herein. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). In additional embodiments, one or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, one or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

The invention also encompasses multivalent and multispecific compositions that bind a target expressed on a leukocyte. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) has a single binding site for a target on a leukocyte so as to direct an immune response to the cell, tissue, or infectious agent of interest. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more antigens and tumor/cancer associated antigens). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In one embodiment, the multivalent and monovalent multispecific composition binds a target expressed on a T cell. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) a target on a T cell so as to juxtapose myeloid cells with the cell, tissue, or infectious agent of interest. In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for (i.e., multivalently binds) a target on a T cell. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for (i.e., monovalently binds) a target on a T cell. In some embodiments the single binding site is an MRD. In other embodiments, the single binding site is an antibody antigen binding domain. In further embodiments, binding of the multivalent and monovalent multispecific composition does not elicit a signal when the composition binds a target on a T cell. In other embodiments, the binding of the multivalent and monovalent multispecific composition does not result in lysis of the T cell expressing the target. In some embodiments, the multivalent and monovalent multispecific composition binds a target selected from: CD2, CD3, CD4, CD8, CD161, a chemokine receptor, CD95, and CCR5. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition is a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In further embodiments, the multivalent and monovalent multispecific composition contains a fusion protein containing one or more peptides that bind to a protein on the surface of a cell, such as a T cell. In additional embodiments, the multivalent and monovalent multispecific composition bind target membrane proximal protein sequences on a cell and inhibit the cross-linking (e.g., multimerization) of the target protein or its associated proteins. In a particular embodiment, the multivalent and monovalent multispecific composition binds to a T cell and inhibits the cross-linking of the cell protein or its associated proteins. For example, in one embodiment, the multivalent and multispecific antibody comprises the amino terminal 27 amino acids of mature CD3 epsilon. In another embodiment, the multivalent and monovalent multispecific composition comprises a fusion protein containing one or more proteins corresponding to the G Domain of a CD3 protein (e.g., CD3 epsilon, CD3 gamma, CD3 alpha (TCRA) or CD3 beta (TCRB). Thus, in some embodiments, the fusion protein comprises a polypeptide having an amino acid sequence selected from: GYYVCYPRGSKPEDANFYLYLR ARVC (SEQ ID NO:21), YLYLRAR (SEQ ID NO:22), YRCNGTDIYKDKESTVQ VHYRMC (SEQ ID NO:23), and DKESTVQVH (SEQ ID NO:24). In additional embodiments, the composition comprises a fusion protein containing one or more proteins corresponding to a portion of the extracellular domain of a CD3 protein (e.g., CD3 epsilon, CD3 gamma, CD3 alpha (TCRA) or CD3 beta (TCRB)) that is able to bind CD3, or a CD3 multimer. Thus, in some embodiments, the fusion protein comprises a portion of a CD3 protein that is able to bind CD3 or a CD3 multimer wherein the portion comprises a CD3 binding fragment of a polypeptide having an amino acid sequence selected from: KIPIEELEDRVFVNCNTSITWVEG TVGTLLSDITRLDLGKRILDPRGIYRCNGTDIY KDKESTVQVHYRMCQSCVELD (human CD3 delta mature ECD, SEQ ID NO:25), QSIKGNHLVKVYDYQEDGSVLLTCDAEAK NITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRM CQNCIELN (human CD3 gamma mature ECD, Ig-like domain highlighted; SEQ ID NO:26), GNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGS DEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVM (human CD3 epsilon mature ECD, Ig-like domain highlighted, SEQ ID NO:27), and QSFGLLDPK (human CD3 zeta mature ECD, SEQ ID NO:28), In alternative embodiments, the fusion protein comprises a chemokine fragment that binds a target on the cell surface. In some embodiments, the chemokine fragment is a portion of a chemokine selected from: CCL20 (LARC/Ckβ4), CCL25 (TECK/Ckβ15), CXCL12 (SDF-1), CXCL13 (BCA-1), CXCL16 (SRPSOX), and CX3CL1 (Fractalkine) In some embodiments, the chemokine fragment is a portion of a chemokine selected from: CCL5 (RANTES), CCL8 (MCP-2), CXCL9 (MIG/CRG-10), CXCL10 (IP-10/CRG-2) and CXCL11 (TAC/IP-9). In some embodiments, the chemokine fragment is a portion of a chemokine selected from CCL3 (MIP-1a) and CCL4 (MIP-113).

In some embodiments, the invention comprises an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds CD3 and contains an amino acid sequence selected from the group consisting of: SEQ ID NO:4329-4492 and 4493, as set forth in Table 22. In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for CD3 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4329-4492 and 4493. In a further embodiment, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds to the same epitope of CD3 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4329-4492 and 4493.

In particular embodiments, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds CD3 and contains an amino acid sequence selected from the group consisting of: IALMCSMHFDEVVWCSPYY (CD31916; SEQ ID NO:4494); PLMLCRHMKHFEYYC WPLA (CD3437; SEQ ID NO:4495); PVICQWTLELQCSPWT (CD3914; SEQ ID NO:4496); ILLECYWEDWRLVCSSLA (CD3434; SEQ ID NO:4497); KGTICWWHLEATCFATS (CD3702; SEQ ID NO:4498); and LREICVKVPYGVVCQRLP (CD31913; SEQ ID NO:4499). In an additional embodiment, the invention encompasses an MRD and/or multivalent and monovalent multispecific composition that competes for CD3 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4494-4498 and 4499. In a further embodiment, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) MRD-containing antibody that binds to the same epitope of CD3 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4494-4498 and 4499.

In particular embodiments, the composition binds a CD3 target selected from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, TCR alpha, TCR beta, the TCR complex, or a heteromeric or homomultimeric combination thereof. In a further embodiment, the composition binds CD3 epsilon. In additional embodiments, the multivalent and monovalent multispecific composition binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen as disclosed herein or otherwise known in the art). In additional embodiments, the multivalent and monovalent multispecific composition has a single binding site for (i.e., monovalently binds) CD3. In further embodiments, the multivalent and monovalent multispecific composition has a single MRD that binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen as disclosed herein or otherwise known in the art). In further embodiments, the multivalent and monovalent multispecific composition has a single antibody antigen binding domain that binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen as disclosed herein or otherwise known in the art). In particular embodiments, the CD3 binding compositions of the invention are not single chain antibodies.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 and a CD3 ortholog from another organism. In additional embodiments, the multivalent and monovalent multispecific composition binds human CD3 and a CD3 ortholog from another primate. In further embodiments, the multivalent and monovalent multispecific composition binds human CD3 and a CD3 ortholog from cynomolgus Monkey or rhesus Monkey. In other embodiments, the multivalent and monovalent multispecific composition binds human CD3 and a CD3 ortholog from a primate selected from Saguinus Oedipus and Callithrix jacchus). In an additional embodiment, the multivalent and monovalent multispecific composition binds human CD3 and a CD3 ortholog from cynomolgus monkey, and a CD3 ortholog from mouse or rat. In particular embodiments, the human CD3 epsilon binding compositions of the invention are not single chain antibodies. In additional particular embodiments, the CD3 binding compositions of the invention are not single chain antibodies.

According to one embodiment, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 epsilon. In a particular embodiment, the, multivalent and monovalent multispecific composition binds human CD3 epsilon protein having the sequence of amino acids 23-207 set forth in NCBI Ref Seq. No. NP_000724. In another embodiment, the multivalent and monovalent multispecific composition binds a polypeptide having the amino acid sequence of QDGNEEMGGITQTPYKVSISGTT VILT (SEQ ID NO:29). In an additional embodiment, the multivalent and monovalent multispecific composition binds a polypeptide having the amino acid sequence of QDGNEEMGGI (SEQ ID NO:30). In a further embodiment, the multivalent and monovalent multispecific composition binds a polypeptide having the amino acid sequence of QDGNEEMGG (SEQ ID NO:31). In particular embodiments, the human CD3 epsilon binding compositions of the invention are not single chain antibodies.

In some embodiments, a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) has a single binding site for CD3 epsilon (i.e., monovalently binds CD3 epsilon) and multiple binding sites for 1, 2, 3, 4, 5 or more different targets (e.g., a B cell or other target disclosed herein). In further embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) competes for binding to CD3 with an antibody selected from: OKT-3, otelixizumab, teplizumab, visilizumab, muromonab, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01. In additional embodiments, an MRD of an MRD-containing antibody competes for binding to CD3 with an antibody selected from: OKT-3, otelixizumab, teplizumab, visilizumab, muromonab X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01. In further embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) competes for binding to CD3 with a CD3 binding composition disclosed in Int. Appl. Pub Nos. WO2004/106380 and WO99/54440; Tunnacliffe et al., Int. Immunol. 1:546-550 (1989); Kjer-Nielsen, PNAS 101:7675-7680 (2004); or Salmeron et al., J. Immunol. 147: 3047-3052 (1991).

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 epsilon and a CD3 epsilon ortholog from another organism. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 epsilon and a CD3 epsilon ortholog from another primate. In additional embodiments, the multivalent and monovalent multispecific composition binds human CD3 epsilon and a CD3 epsilon ortholog from cynomolgus monkey or rhesus monkey. In additional embodiments, the multivalent and monovalent multispecific composition binds human CD3 epsilon and a CD3 epsilon ortholog from a primate selected from Saguinus Oedipus and Callithrix jacchus. In an additional embodiment, the multivalent and monovalent multispecific composition binds human CD3 epsilon and a CD3 epsilon ortholog from cynomolgus monkey, and a CD3 epsilon ortholog from mouse or rat. In particular embodiments, an MRD of the multivalent and monovalent multispecific composition binds CD3 epsilon.

In another embodiment the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 delta. In a particular embodiment, the, multivalent and monovalent multispecific composition binds human CD3 delta having the sequence of amino acids 22-171 set forth in NCBI Ref. Seq. No. NP_000723. In particular embodiments, an MRD of the multivalent and monovalent multispecific composition binds CD3 delta. In other embodiments, an antibody antigen binding domain of the multivalent and monovalent multispecific composition binds CD3 delta. In particular embodiments, the human CD3 epsilon binding compositions of the invention are not single chain antibodies.

In an additional embodiment, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 gamma protein having the sequence of amino acids 23-182 set forth in NCBI Ref. Seq. No. NP_000064. In particular embodiments, an MRD of the multivalent and monovalent multispecific composition binds gamma. In particular embodiments, an MRD of the multivalent and monovalent multispecific composition binds CD3 gamma. In other embodiments, an antibody antigen binding domain of the multivalent and monovalent multispecific composition binds CD3 gamma. In particular embodiments, the human CD3 gamma binding compositions of the invention are not single chain antibodies.

In an additional embodiment, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD3 zeta protein having the sequence of amino acids 22-164 set forth in NCBI Ref. Seq. No. NP_932170. In particular embodiments, an MRD of the multivalent and monovalent multispecific composition binds CD3 zeta. In other embodiments, an antibody antigen binding domain of the multivalent and monovalent multispecific composition binds CD3 zeta. In particular embodiments, the human CD3 zeta binding compositions of the invention are not single chain antibodies.

The invention also encompasses multivalent and multispecific compositions that bind a target expressed on a natural killer cell. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) a target on a natural killer cell. In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for (i.e., multivalently binds) a target on a natural killer cell. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for (i.e., monovalently binds) a target on a natural killer cell. In some embodiments the single binding site is an MRD. In other embodiments, the single binding site is an antibody antigen binding domain. In further embodiments, binding of the multivalent and monovalent multispecific composition does not elicit a signal when the composition binds a target on a natural killer cell. In some embodiments, the multivalent and monovalent multispecific composition binds a target selected from: KLRD1, KLRK1, KLRB1, 2B4 (CD244), KIR2D4, KIR2D5, and KIR3DL1. In other embodiments, the multivalent and monovalent multispecific composition binds a target selected from: CD56, CD2, and CD161. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In a particular embodiment, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds NKGD2 and contains an amino acid sequence selected from the group consisting of: FSLFCYNVHGWWECFPVY (NKG1; SEQ ID NO:4500); SLTWCMVEKLHYWVICDRVA (NKG4; SEQ ID NO:4501); ILISCQEQWPVFQCYAVR (NKG7; SEQ ID NO:4502); HEEDCYWILYQHCPRAT (NKG9; SEQ ID NO:4503); LEHLCTTYPMWFHCPEGA (NKG12 SEQ ID NO:4504); SGLVCFSRFDWWECVWTS (NKG14; SEQ ID NO:4505); SLTRSRYQRFHYWVICDRVA (NKG15; SEQ ID NO:4506); and YTVACYYGVDQY WMCFSNS (NKG17; SEQ ID NO:4507). In an additional embodiment, the invention encompasses an MRD and/or multivalent and monovalent multispecific composition that competes for NKGD2 binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4500-4506 and 4507. In a further embodiment, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) MRD-containing antibody that binds to the same epitope of NKGD2 as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4500-4506 and 4507.

In some embodiments, the invention comprises an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds CD319 (CRACC) and contains an amino acid sequence selected from the group consisting of: WRLDCWEHHEWDFWCWAHG (SEQ ID NO:4508); SLYECWRVFVSFPRCPDGS (SEQ ID NO:4509); LYLLCEHVHDKHWECGSWL (SEQ ID NO:4510); IHLRCTYVEPLYML CSPYA (SEQ ID NO:4511); TMMVECYMGYCYPTVF (SEQ ID NO:4512); VLLRCQYVGV SHIKCKSVD (SEQ ID NO:4513); LVLECFLIDAWYMKCHTTG (SEQ ID NO:4514); LRL YCIPVDHTFFKCTLYG (SEQ ID NO:4515); YCIYRCQVQQCWMFPA (SEQ ID NO:4516); WHIACWEMRDVHWYVCEFFV (SEQ ID NO:4517); RVLQCKWVSSEYFQCVETS (SEQ ID NO:4518); VVVECFQVKEMYWSCRPAV (SEQ ID NO:4519); and YLMYCTALEYPYF QCRQMV (SEQ ID NO:4520). In another embodiment, the invention encompasses an MRD and/or MRD-containing antibody that competes for CRACC binding with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4508-4519 and 4520. In a further embodiment, the invention encompasses an MRD and/or a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that binds to the same epitope of CRACC as a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:4508-4519 and 4520.

In specific embodiments, the multivalent and monovalent multispecific composition binds CD2. According to one embodiment, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD2. In a particular embodiment, the multivalent and monovalent multispecific composition binds human CD2 protein having the sequence of amino acids 25-209 set forth in NCBI Ref. Seq. No. NP_001758. In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for CD2. In some embodiments the single binding site is an MRD. In other embodiments, the single binding site is an antibody antigen binding domain. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for CD2. In further embodiments, binding of the multivalent and monovalent multispecific composition to CD2 does not elicit a signal by the cell on which CD2 is expressed. In additional embodiments, the multivalent and monovalent multispecific composition binds CD2 and 1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen as disclosed herein or otherwise known in the art). In particular embodiments, the CD2 binding compositions of the invention are not single chain antibodies.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds human CD2 and a CD2 ortholog from another organism. In additional embodiments, the multivalent and monovalent multispecific composition binds human CD2 and a CD2 ortholog from another primate. In further embodiments, the multivalent and monovalent multispecific composition binds human CD2 and a CD2 ortholog from cynomolgus monkey or rhesus monkey.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds a target on a myeloid cell. In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds (1) a target on a cell, tissue, or infectious agent of interest (e.g., a tumor antigen on a tumor cell) and (2) a target on an immune accessory cell (e.g., myeloid cell) so as to juxtapose myeloid cells with the cell, tissue, or infectious agent of interest. In some embodiments, the multivalent and monovalent multispecific composition has multiple binding sites for (i.e., multivalently binds) a target on a myeloid cell. In other embodiments, the multivalent and monovalent multispecific composition has a single binding site for (i.e., monovalently binds) a target on an accessory cell (e.g., myeloid cell). In some embodiments the single binding site is an MRD. In other embodiments, the single binding site is an antibody antigen binding domain. In further embodiments, binding of the multivalent and monovalent multispecific composition does not elicit a signal when the composition binds a target on a myeloid cell. In some embodiments, the multivalent and monovalent multispecific composition binds an Fc gamma receptor selected from CD16 (i.e., Fc gamma Rill), CD64 (i.e., Fc gamma RI), and CD32 (i.e., Fc gamma RII). In particular embodiments, the multivalent and monovalent multispecific composition binds CD64 (i.e., Fc gamma RI). In some embodiments, the multivalent and monovalent multispecific composition binds a target selected from, MHC class 2 and its invariant chain, TLR1, TLR2, TLR4, TLR5 and TLR6. In additional embodiments, the multivalent and monovalent multispecific composition binds at least 2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of interest. According to some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the multivalent and monovalent multispecific composition are located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are a tumor antigen (e.g., tumor antigens and tumor/cancer associated antigens). In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the immune system. In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent and monovalent multispecific composition are associated with a disease or disorder of the skeletal system (e.g., osteoporosis), cardiovascular system, nervous system, or an infectious disease.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds a target of interest on a cancer cell. In additional embodiments, the multivalent and monovalent multispecific composition binds a target of interest on an immune cell. In further embodiments, the multivalent and monovalent multispecific composition binds a target of interest on a diseased cell. In other embodiments, the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds a target of interest on an infectious agent (e.g., a bacterial cell or a virus).

In further embodiments, the invention encompasses a method of treating a disease or disorder by administering to a patient in need thereof, a therapeutically effective amount of a multivalent and monovalent multispecific composition of the invention. Particular embodiments are directed to a method of treating a disease or disorder by administering to a patient in need thereof, a therapeutically effective amount a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that has a single binding site for a target (i.e., that monovalently binds a target). In some embodiments, the administered multivalent and monovalent multispecific composition has a single binding site for a target on a leukocyte, such as a T-cell (e.g., CD3). In additional embodiments, the administered multivalent and monovalent multispecific composition has a single binding site for a target on a leukocyte, such as a T-cell (e.g., CD3) and multiple binding sites for (i.e., is capable of multivalently binding) a target located on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell).

In further embodiments, the invention is directed to treating a disease or disorder by administering to a patient a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that has a single binding site for a target (i.e., that monovalently binds a target) and multiple binding sites for 1, 2, 3, 4, 5 or more different targets.

In additional embodiments, the invention is directed to treating a disease or disorder by administering to a patient in need thereof, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that has a single binding site for CD3 (e.g., CD3 epsilon) that monovalently binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more different targets.

According to some embodiments, the tumor cell is from a cancer selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer.

In some embodiments, the MRD(s) and the antibody in the MRD-containing antibody are antagonists of their respective targets. In other embodiments, the MRD(s) and the antibody in the MRD-containing antibody are agonists of their respective target. In yet other embodiments, at least one of the MRDs in the MRD-containing antibody is an antagonist of its target molecule and the antibody is an agonist of its target molecule. In yet another embodiment, at least one of the MRDs in the MRD-containing antibody is an agonist of its target molecule, and the antibody is an antagonist of its target molecule.

In some embodiments, both the MRD(s) and the antibody in the MRD-containing antibody bind to soluble factors. In some embodiments, both the MRD(s) and the antibody in the MRD-containing antibody bind to cell surface molecules. In some embodiments, at least one MRD in the MRD-containing antibody binds to a cell surface molecule and the antibody in the MRD-containing antibody binds to a soluble factor. In some embodiments, at least one MRD in the MRD-containing antibody binds to a soluble factor and the antibody in the MRD-containing antibody binds to a cell surface molecule.

An improved multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) that specifically binds a desired target or targets can also be prepared based on a previously known MRD or multivalent and monovalent multispecific composition (e.g., MRD-containing antibody). For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, 50-100, 100-150 or more than 150 amino acid substitutions, deletions or insertions can be introduced into an MRD or multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) sequence and the resulting MRD or multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) can be screened for binding to the desired target or targets, for antagonizing target activity, or for agonizing target activity as described in the examples or using techniques known in the art.

Additional peptide sequences may be added, for example, to enhance the in vivo stability of the MRD or affinity of the MRD for its target.

In certain embodiments, the binding of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to its target (e.g., a cell) is enhanced compared to the binding of the MRD alone, the antibody alone, and/or a combination of the MRD and antibody. In some embodiments, the binding is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold improved.

In addition, in some embodiments, the binding of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to a target (e.g., a cell or a molecule containing multiple epitopes) expressing both the MRD target and the antibody target is enhanced compared to the binding of the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to a target (e.g., a cell or a molecule containing multiple epitopes) expressing only the MRD target or only the antibody target. In some embodiments, the binding is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold improved. This increased avidity can enable multivalent and multispecific compositions (e.g., MRD-containing antibodies) to bind to targets that have previously been difficult to target, e.g., G-protein coupled receptors and carbohydrate molecules.

In addition, in some embodiments, the binding of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to an MRD target is enhanced in a region (e.g., of the body) where the antibody target is localized compared to a region where the antibody target is not expressed or is expressed at a lower level. In some embodiments, the binding of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to an antibody target is enhanced in a region (e.g., of the body) where the MRD target is localized compared to a region where the MRD target is not expressed or is expressed at a lower level. In some embodiments, the binding is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold improved.

In preferred embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) retains particular activities of the parent antibody. Thus, in certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is capable of inducing complement dependent cytotoxicity. In certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is capable of inducing antibody dependent cell mediated cytotoxicity (ADCC). In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is capable of inducing apoptosis. In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is capable of reducing tumor volume. In additional embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are capable of inhibiting tumor growth.

In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) shows improved activity or pharmacodynamic properties compared to the corresponding antibody without the attached MRD. Thus, in certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has greater avidity than the corresponding antibody without the attached MRD. In other embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) results in increased receptor aggregation compared to the corresponding antibody without the attached MRD. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) antagonizes target activity to a greater extent than the corresponding antibody without the attached MRD. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) agonizes target activity to a greater extent than the corresponding antibody without the attached MRD. In another embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has an improved pharmacodymamic profile than the corresponding antibody without the attached MRD.

In another embodiment, the MRD-containing antibody has a greater therapeutic efficacy than the corresponding antibody without the attached MRD.

In other embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) have one or more of the following effects: inhibit proliferation of tumor cells, reduce the tumorigenicity of a tumor, inhibit tumor growth, increase patient survival, trigger cell death of tumor cells, differentiate tumorigenic cells to a non-tumorigenic state, or prevent metastasis of tumor cells.

In certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is at least as stable as the corresponding antibody without the attached MRD. In certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is more stable than the corresponding antibody without the attached MRD. MRD-antibody stability can be measured using methods known to those in the art, including, for example, ELISA techniques. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is stable in whole blood at 37° C. for at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, or at least about 100 hours.

In certain embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has at least the same affinity for Fc receptors as the corresponding parent antibody. In other nonexclusive embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has at least the same affinity for complement receptors as the corresponding parent antibody. In other nonexclusive embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has at least the same half-life as the corresponding parent antibody. In other embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) can be expressed at levels commensurate with the corresponding parent antibody.

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has an increased affinity for Fc receptors compared to the corresponding parent antibody. In other nonexclusive embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has an increased affinity for complement receptors compared to the corresponding parent antibody. In other nonexclusive embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) has an increased half-life compared to the corresponding parent antibody. In other embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) can be expressed at increased levels compared to that of the corresponding parent antibody.

Immunoconjugates (MRD-Containing Antibody Drug Conjugates)

The use of antibody-drug conjugates for the local delivery of cytotoxic agents, allows targeted delivery of the drug to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet pages 603-05 (1986); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.s), pp. 475-506) (1985)).

In additional embodiments, the invention encompasses a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) that is covalently or otherwise associated with a cytotoxic agent (payload) (i.e., as multivalent and monovalent multispecific-cytoxic agent complexes (e.g., MRD-containing antibody-cytoxic agent complexes). According to some embodiments, the cytoxic agent is covalently attached to a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) by a linker. According to some embodiments, the linker attaching the multivalent and monovalent multispecific composition and the cytotoxic agent is cleavable by a protease. In additional embodiments, the cytotoxic agent is a chemotherapeutic agent, growth inhibitory agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a radioactive isotope (i.e., a radioconjugate) or a prodrug. Methods of using immunoconjugates (MRD-containing Antibody drug conjugates) are also encompassed by the invention.

Cytotoxic agents that may be covalently or otherwise associated with multivalent and multispecific compositions (e.g., an MRD-containing antibody) include, but are not limited to any agent that is detrimental to (e.g., kills) cells. Cytotoxins useful in the compositions and methods of the invention include, inter alia, alkylating agents, intercalating agents, antiproliferative agents, anti-mititotic agents, tubulin binding agents, vinca alkaloids, enediynes, trichothecenes, podophyllotoxins or podophyllotoxin derivatives, the pteridine family of drugs, taxanes, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin, dolastatins (e.g., dolastatin 10, dolastatin 11, and dolastatin 15)), topoiosomerase inhibitors, and platinum complex chemotherapeutic agents (e.g., cis-platinum).

In some embodiments, compositions of the invention include a cytoxic agent that is a tubulin depolymerizing agent. Thus, in some embodiments, compositions of the invention include an auristatin or an auristatin derivative or analog. In one embodiment, compositions of the invention contain monomethyl auristatin E (MMAE). In another embodiment, compositions of the invention contain monomethyl auristatin F (MMAF). In additional embodiments, an immunoconjugate composition of the invention contains dolastatin or a dolastatin peptidic analog or derivative, e.g., an auristatin (see, e.g., U.S. Pat. Nos. 5,635,483, 5,780,588, and 5,663,149).

In additional embodiments, compositions of the invention include a maytansinoid molecule. Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Methods of making maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064, 6,441,163 and European Pat. EP 0 425 235 B1; each of which is herein incorporated by reference in its entirety.

Thus, in some embodiments, the cytotoxin is a maytansinoid or a maytansinoid derivative or analog. Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines. Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al PNAS 99:7968-7973 (2002)), or maytansinol and maytansinol analogues can be prepared synthetically according to known methods.

In particular embodiments compositions of the invention include the maytansinoid DM1 (N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine). In other particular embodiments compositions of the invention include the maytansinoid DM2. In additional embodiments, compositions of the invention include the maytansinoid DM3 (N(2′)-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine) or DM4 (N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).

In some embodiments, compositions of the invention include a cytoxic agent that is an alkylating agent. In particular embodiments, the cytotoxic agent is selected from mechlorethamine, thiotepa, thioepa chlorambucil, melphalan, carmustine (BSNU), BCNU lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, and streptozoicin.

In other embodiments, compositions of the invention include a cytoxic agent that is an antimetabolite. In particular embodiments, the cytotoxic agent is selected from methotrexate, dichloromethotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and 5-fluorouracil decarbazine.

In additional embodiments, the multivalent and multispecific composition-drug conjugate (e.g., MRD-containing antibody-drug conjugate) is capable of producing double-stranded DNA breaks. In further embodiments, the MRD-containing antibody-drug conjugate contains a member of the calicheamicin family of antibiotics capable of producing double-stranded DNA breaks at sub-picomolar concentrations. In further embodiments, a multivalent and multispecific composition-drug conjugate (e.g., MRD-containing antibody-drug conjugate) contains calicheamycin. For the preparation of conjugates of the calicheamicin family, see e.g., U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which can be contained in the multivalent and multispecific composition-drug conjugate (e.g., MRD-containing antibody-drug conjugate) of the invention include, but are not limited to, gamma₁ ^(I), alpha₂ ^(I), alpha₃ ^(I), N-acetylamma₁ ^(I), PSAG and theta₁ ^(I) (Hinman et al., Cancer Research 53:3336-3342 (1993), and Lode et al., Cancer Research 58:2925-2928 (1998).

In other embodiments, multivalent and multispecific composition-drug conjugate (e.g., MRD-containing antibody-drug conjugate) compositions of the invention include a cytoxic agent selected from adriamicin, doxorubicin, mitomycin C, busulfan, cytoxin, chlorambucil, etoposide, etoposide phosphate, CC-1065, duocarmycin, KW-2189, CC1065, taxotere (docetaxel), methopterin, aminopterin, topotecan, camptothecin, porfiromycin, bleomycin, teniposide, esperamicins, mithramycin, anthramycin (AMC), fludarabine, tamoxifen, taxotere (docetaxel), cytosine arabinoside (Ara-C), adenosine arabinoside, cisplatin, carboplatin, cis-dichlorodiamine platinum (II) (DDP) cisplatin, chloroquine, cyclosporin A, docetaxel, paclitaxel, taxol, vinorelbine, vindesine, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, ifosfamide, cyclophosphamide, tenoposide, carminomycin, porfiromycin, dihydroxy anthracin dione, mitoxantrone, mithramycin, dactinomycin, actinomycin D, puromycin 1-dehydrotestosterone, adriamycin, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, epithiolone, QFA, combretastatin, combretastatin A4 phosphate, vinblastine, vincristine, colchicine, geldanamycin, doxorubicinchlorambucil, Auristatin F phenylene diamine (AFP)), monomethylauristatin, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296) or a derivative or analog thereof and derivatives and analog thereof.

Additional suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985). Moreover, for further discussion of types of cytotoxins, linkers and other methods that can be use or routinely adapted to conjugate therapeutic agents to the MRD-comprising antibody complex, see e.g., Intl. Appl. Publ. WO2007/059404; Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan et al., Curr. Opin. Investig. Drugs 3:1089-1091 (2002); and Senter et al., Adv. Drug Deliv. Rev. 53:247-264 (2001), each of which is hereby incorporated by reference in its entirety.

Cytotoxin chemotherapeutic agents that can be used in the immunoconjugates of the invention (e.g., multivalent and multispecific composition-drug conjugates such as MRD-containing antibody-drug conjugates) include poisonous lectins and plant or other toxins (e.g., ricin, abrin, modeccin, botulina, and diphtheria toxins). It is envisioned that multiple copies of a toxin or combinations of various toxins can optionally be coupled to a multispecific and multivalent composition of the invention (e.g., an MRD-containing antibody) thereby providing additional cytotoxicity. Enzymatically active toxins and fragments thereof that can be used in compositions of the invention include, but are not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), Pseudomonas exotoxin, Pseudomonas endotoxin, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, ribonuclease, DNase I, Staphylococcal enterotoxin-A, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, for example, Pastan et al., Cell 47:641 (1986), Goldenberg et al., Cancer Journal for Clinicians 44:43 (1994) and Intl Appl. Publ. Nos. WO93/21232 and WO93/21232, each of which is herein incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, “The Peptides”, volume 1, pp. 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. Nos. 5,635,483 and 5,780,588; Pettit et al., J. Am. Chem. Soc. 111:5463-5465 (1989); Pettit et al., Anti-Cancer Drug Design 13:243-277 (1998); Pettit et al., Synthesis 719-725 (1996); Pettit et al., J. Chem. Soc. Perkin Trans. 15:859-863 (1996); and Doronina et al., Nat. Biotechnol 21(7):778-784 (2003).

According to some embodiments, the compositions of the invention comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated multivalent and multispecific compositions (e.g., MRD-containing antibodies). Examples include At²¹¹, I¹³¹, I¹²⁵, Y.⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintiographic studies, for example tc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels can be incorporated in the conjugate using techniques known in the art. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al Biochem. Biophys. Res. Commun. 80: 49-57 (1978)) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes in detail other methods that can be routinely applied to label the compositions of the invention.

A linker can be a “cleavable linker,” facilitating release of a drug in the cell. For example, an acid-labile linker (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020, U.S. Pat. Appl. Publ. No. 20110293513) can be used. Thus, the invention encompasses multivalent and multispecific compositions containing one or more linkers that can contain any of a variety of groups as part of its chain that will cleave in vivo, e.g., in a cell, at a rate which is enhanced relative to that of constructs that lack such groups. Also provided are conjugates of the linker arms with therapeutic and diagnostic agents. The linkers are useful to form prodrug analogs of therapeutic agents and to reversibly link a therapeutic or diagnostic agent (e.g., a cytotoxin or MRD) to a targeting agent, a detectable label, or a solid support. The linkers can be stable in plasma so as not to release an MRD or cytotoxic agent. In the case of cytotoxins the linkers can be stable in plasma and labile once internalized so as to release the cytotoxin in an active form.

MRDs and/or cytotoxic agents are optionally attached to one another or to the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) of the invention with a linker as described herein or otherwise known in the art. Conjugates of the MRD-containing antibody with an MRD or a cytotoxic agent can be made using a variety of bifunctional protein coupling agents known in the art, including, but not limited to, coupling agents containing a group selected from: 6-maleimidocaproyl (MC), maleimidocaproyl-polyethylene glycol (“MC(PEG)6-OH” (amenable to attachment to antibody cysteines)), maleimidopropanoyl (MP), MPBH, valine-citrulline (val-cit (exemplary dipeptide in a protease cleavable linker)), methyl-valine-citrulline (“Me-Val-CitN,” a linker in which a peptide bond has been modified to prevent its cleavage by cathepsin B) alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB (an example of a “self immolative” linker component)), valine-citrullin-p-aminobenzyloxycaronyl (“vc-PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), LC-SMCC, N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), IT (iminothiolane), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), ethyleneoxy-CH₂CH₂O— as one or more repeating units (“EO” or “PEO”), BMPS, EMCS, GMBS, HBVS, MBS, SBAP, SIA, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SMCC, sulfo-SIAB, sulfo-SMPB, SVSB (succinimidyl-(4-vinylsulfone) benzoate), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Additional linker components are known in the art and some are described herein.

In some embodiments, the multivalent and monovalent multispecific composition is covalently attached to a cytotoxic agent via a linker at 1-5, 5-10, 1-10, or 1-20 sites on the multivalent and multispecific composition. According to additional embodiments, the multivalent and monovalent multispecific composition is covalently attached to a cytotoxic agent via a linker at more than 2, 5 or 10 sites on the multivalent and multispecific composition.

In additional embodiments, the multivalent and monovalent multispecific composition (e.g., MRD containing antibody) complex is associated with a prodrug. Prodrug synthesis, chemical linkage to antibodies, and pharmacodynamic properties are known in the art and can routinely be applied to make and use multivalent and multivalent compositions of the invention that contain prodrugs, such as, MRD-containing antibody-prodrug compositions. See, e.g., Intl. Publ. No. WO96/05863 and in U.S. Pat. No. 5,962,216, each of which is herein incorporated by reference in its entirety.

Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be made, e.g., by recombinant techniques or peptide synthesis. A recombinant DNA molecule can comprise regions encoding the antibody and cytotoxic portions of the conjugate either adjacent to one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

The multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) composition of the invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to multivalent and monovalent multispecific compositions (e.g., MRD containing antibodies) for use diagnostically or therapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹, yttrium⁹⁰, and lutetium¹⁷⁷. Methods for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™. (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the MRD-containing antibodies of the invention.

Methods for the conjugation of linker-drug moieties to cell-targeted proteins such as antibodies are known in the art and include those described for example, in U.S. Pat. Nos. 5,208,020 and 6,441,163; Intl. Appl. Publ. Nos. WO2005037992, WO2005081711, and WO2006/034488, each of which is herein incorporated by reference in its entirety. See, also e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen et al., Nat. Rev. Cancer 2:750-763 (2002); Pastan et al., Curr. Opin. Investig. Drugs 3:1089-1091 (2002); and Senter et al., Adv. Drug Deliv. Rev. 53:247-264 (2001), the contents of each of which is herein incorporated by reference in its entirety.

In some embodiments, a multivalent and monovalent multispecific composition of the invention comprising a cytotoxic agent (e.g., an MRD-containing antibody-cytotoxic agent conjugate) and may generally be referred to herein as an immunoconjugate. In some embodiments, an immunoconjugate of the invention binds a cell surface target that is internalized into the cell. In further embodiments, the binding of an immunoconjugate of the invention (e.g., an MRD-containing antibody-cytotoxic agent conjugate) to a cell surface target results in the internalization of the immunoconjugate into the cell in vitro. In further embodiments, the binding of immunoconjugate to a cell surface target results in the internalization of the composition into the cell in vivo. Methods for treating a patient described herein can comprise: administering to the patient a therapeutically effective amount of an immunoconjugate (e.g., a multivalent and monovalent multispecific composition of the invention comprising a cytotoxic agent, such as an MRD-containing antibody-cytotoxic agent conjugate) that comprises a cytotoxic agent and binds a target that is internalized into a cell. In some embodiments, the immunoconjugate comprises a cytotoxic agent disclosed herein. In additional embodiments, the immunoconjugate comprises a cytotoxic agent selected from an alkylating agent, antiproliferative agent, tubulin binding agent, vinca alkaloid, enediyne, podophyllotoxin, podophyllotoxin derivative, a member of the pteridine family of drugs, taxane, a dolastatin, topoiosomerase inhibitor, or a platinum complex chemotherapeutic agent. In further embodiments, the cytoxic agent is a maytansinoid or a maytansinoid derivative or analog. In specific embodiments the cytoxic agent is the maytansinoid DM1, DM2, or DM3. In additional embodiments, the cytotoxic agent is auristatin or an auristatin derivative or analog. In specific embodiments the cytoxic agent is MMAE or MMAF. The cytotoxic agents are optionally attached to the other components of the immunoconjugate by a linker. In some embodiments the cytotoxic agent is attached to the other components of the immunoconjugate by an enzyme cleavable linker. In additional embodiments, the cytotoxic agent is attached to the other components of the immunoconjugate by an acid-labile linker.

In further embodiments, the cytoxic agent of an immunoconjugate of the invention has a free drug potency of less than 10⁻⁷M, 10⁻⁸M, or 10⁻⁹M. In additional embodiments, the cytoxin has a free drug potency of 10⁻⁸ to 10⁻¹¹ M.

In some embodiments, a target bound by the immunoconjugate is selected from CD19, CD22, CD30, CD33, CD56, CD70, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, PSMA, EphA2, ErbB2 (CD340), SLC44A4, MN (carbonic anhydrase IX), GPNMB (glycoprotein non-metastatic melanoma protein), Cripto, and αV integrin. In additional embodiments, a target bound by the immunoconjugate is selected from CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD25, TNFRSF5 (CD40), CD64, CD74, CD79, CD105, CD174, CD205, CD227, CD326, CD340, MUC16, EGP-1, EGP-2, EGF receptor (ErbB1), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor, Ga 733, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF), IFN-gamma, IFN-alpha, IFN-beta, IL2R, IL4R, IL6R, IL13R, IL15R, IL17R, IL18R, IL2, IL6, IL8, IL12, IL15, IL17, IL18, IL25, IP-10, IGF-1R, Ia, HM1.24, HCG, HLA-DR, ED-B, TMEFF2, EphB2, FAP (fibroblast activation protein), mesothelin, EGFR, TAG-72, GD2 (encoded by the B4GALNT1 gene), and 5T4.

In additional embodiments, a target bound by the immunoconjugate is a myeloid and hematopoietic target selected from CD33, CD64, TNFRSF5 (CD40), CD56, and CD138. In further embodiments, a target bound by the immunoconjugate is a carcinoma target selected from EpCam, GD2, EGFR, CD74, CD227, CD340, MUC16, GD2, GPNMB, PSMA, crypto, TMEFF2, EphB2, 5t4, mesothelin, TAG-72, and MN.

In other embodiments, a target bound by the immunoconjugate is a B cell target selected from CD19/CD21, CD20, CD22, TNFRSF5 (CD40), CD70, CD79a, CD79b, and CD205. In additional embodiments, a target bound by the immunoconjugate is a T cell target selected from CD25, CD30, TNFRSF5 (CD40), CD70, and CD205. In further embodiments, a target bound by an endothelial cell target selected from CD105, the stromal cell target FAP, and the vascular target ED-B.

In additional embodiments, an immunoconjugate of the invention binds to a cell surface tumor antigen and a second target that is associated with an escape pathway for resisting chemotherapy. In some embodiments, the cell surface tumor antigen is a member selected from EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC.

In further embodiments, an immunoconjugate binds P-glycoprotein (encoded by MDR1) and a cell surface antigen. In particular embodiments, the cell surface antigen is a tumor antigen. In further embodiments, the tumor antigen is a member selected from: EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC. In particular embodiments, the immunoconjugate competes for CD30 binding with SGN-35 (brentuximab). In additional embodiments, the immunoconjugate competes for ErbB2 binding with trastuzumab.

In further embodiments, an immunoconjugate binds MRP (Multidrug-Resistance associated Protein) and a cell surface antigen. In particular embodiments, the cell surface antigen is a tumor antigen. In further embodiments, the tumor antigen is a member selected from: EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC. In particular embodiments, the immunoconjugate competes for CD30 binding with SGN-35 (brentuximab). In additional embodiments the immunoconjugate competes for ErbB2 binding with trastuzumab.

Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

The following embodiments are further provided for any of the above immunoconjugates. In one embodiment, an immunoconjugate has in vitro or in vivo cell killing activity. In one embodiment, the linker is attached to the antibody through a thiol group on the antibody. In one embodiment, the linker is cleavable by a protease. In one embodiment, the linker comprises a val-cit dipeptide. In one embodiment, the linker comprises a p-aminobenzyl unit. In one embodiment, the p-aminobenzyl unit is disposed between the drug and a protease cleavage site in the linker. In one embodiment, the p-aminobenzyl unit is p-aminobenzyloxycarbonyl (PAB). In one embodiment, the linker comprises 6-maleimidocaproyl. In one embodiment, the 6-maleimidocaproyl is disposed between the antibody and a protease cleavage site in the linker. The above embodiments may occur singly or in any combination with one another.

The MRD-containing antibody of the present invention may also be conjugating to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see e.g., WO81/01145) to an active anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No. 4,975,278 the contents of which are herein incorporated by reference in its entirety. The enzyme component of the immunoconjugate is preferably capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form. See, for example, Pastan et al., Cell, 47:641 (1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43 (1994). Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO93/21232.

In some embodiments, the multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies) are conjugated to a radioisotope, such as, ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re using anyone of a number of well-known chelators or direct labeling. In other embodiments, the MRD-containing antibody is coupled to drugs, prodrugs or lymphokines such as, interferon. Compositions of the invention can be labeled with ligand reagents that bind, chelate or otherwise complex a radioisotope metal where the reagent is reactive with the engineered cysteine thiol of the antibody, using techniques known in the art such as, those described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991). Chelating ligands which may complex a metal ion and that may have use in the compositions and methods of the invention include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.). Radionuclides can be targeted via complexation with the antibody-drug conjugates of the invention (Wu et al Nature Biotechnology 23(9): 1137-1146 (2005)). Linker reagents such as, DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropylchloroformate (Aldrich), following the procedure of Axworthy et al., Proc. Natl. Acad. Sci. USA 97(4):1802-1807 (2000)). DOTA-maleimide reagents react with the free cysteine amino acids of the cysteine engineered antibodies and provide a metal complexing ligand on the antibody (Lewis et al., Bioconj. Chem. 9:72-86 (1998)). Chelating linker labeling reagents such as, DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) are commercially available (Macrocyclics, Dallas, Tex.).

Conjugates of the multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies) and cytotoxin can routinely be made using a variety of bifunctional protein-coupling agents such as, N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as, dimethyl adipimidate HCL), active esters (such as, disuccinimidyl suberate), aldehydes (such as, glutareldehyde), bis-azido compounds (such as, bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as, bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as, tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as, 1,5-difluoro-2,4-dinitrobenzene). In specific embodiments, the toxin is conjugate to an MRD-containing antibody through an enzyme-cleavable linker system (e.g., such as, that present in SGN-35). Conjugates of an MRD-containing antibody and one or more small molecule toxins, such as, a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used.

In some embodiments, the MRD-containing antibody can be complexed, or have MRDs that bind with other immunologically active ligands (e.g., chemokines, cytokines, and antibodies or fragments thereof) wherein the resulting molecule binds to the neoplastic cell or other target as well as the chemokine, cytokine, or an effector cell such as, a T cell. In certain embodiments, these conjugates can be generated as fusion proteins. The enzymes of this invention can be covalently bound to the antibody by techniques well-known in the art such as, the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques known in the art.

In some embodiments, the N-terminus or C-terminus of the antibody to which an MRD is operably linked in the MRD-antibody fusions is truncated. In preferred embodiments, this truncation does not prevent or reduce the ability of the antibody to bind to its target antigen via its antigen binding domain. In other embodiments, the truncation does not prevent or reduce Fc effector function, half-life and/or ADCC activity. In other embodiments, MRDs are attached in the terminal region of the antibody chain. More particularly, in certain embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues of the C-terminal amino acid of the heavy chain. In other embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues of the C-terminal amino acid of the light chain. In additional embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues of the N-terminal amino acid of the heavy chain. In other embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues of the N-terminal amino acid of the light chain. Thus, for example, a MRD that is linked to the N-terminal end of the heavy chain can be linked to the first, second, third, fourth, fifth, or tenth amino acid of the N-terminal chain of the heavy chain. For example, an MRD-antibody fusion containing an MRD linked to the N-terminal of the heavy chain may contain amino acids 1-3 of the heavy chain sequence linked to the MRD, which is linked to amino acid 4 of the heavy chain sequence.

In certain embodiments, one or more MRDs are attached to an antibody at locations other than the termini of the antibody light and heavy chains. The MRD can be attached to any portion of the antibody that does not prevent the ability of the antibody to bind its target. Thus, in some embodiments, the MRD is located outside the antibody combining site. For example, the MRD can be located within a heavy chain sequence or within a light chain sequence. By way of example only, the MRD can be located between the Fc domain and the hinge region, between the hinge region and the CH1 domain of the heavy chain, between the CH1 domain and the variable region of the heavy chain, or between the constant region and the variable region of the light chain.

Angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathways have been observed to provide at best transitory therapeutic benefits followed by restoration of tumor growth and progression due to an apparent ability of angiogenic tumors to adapt to the presence of these inhibitors. Without being bound by theory, it is believed that the multivalent and multispecific properties of multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind an angiogenesis target provide these compounds with an ability to extend anti-angiogenic therapeutic benefits beyond those observed from for example, conventional monoclonal antibody therapies by binding multiple distinct angiogenesis related targets and thereby disrupting resistance mechanisms available to the angiogenic tumor.

In one embodiment, an MRD-containing antibody binds 2 or more targets selected from: VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIc, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TIE2, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGF and 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is bevacizumab. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGF and 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is bevacizumab.

In one embodiment, an MRD-containing antibody binds VEGF (i.e., VEGFA) and additionally binds an angiogenic target selected from: VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL-6, IL-8, IL-18, HGF, TIE2, PDGFA, P1GF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGF and 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is bevacizumab. In additional embodiments, the antibody component of the MRD-containing antibody competes for VEGF binding with bevacizumab.

In one embodiment, an MRD-containing antibody binds TNF alpha and additionally binds a target selected from: Te38, IL-12, IL-12p40, IL-13, IL-15, IL-17, IL-18, IL-1beta, IL-23, MIF, PEG2, PGE4, VEGF, TNFSF11 (RANKL), TNFSF13B (BLYS), GP130, CD-22, and CTLA-4. In another embodiment, an MRD-containing antibody binds TNF alpha, IL6, and TNFSF13B (BLYS). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNF and 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNF. In further embodiments, the antibody component of the MRD-containing antibody is adalimumab, certolizumab, golimumab or AME-527. In additional embodiments, the antibody component of the MRD-containing antibody competes for TNF binding with adalimumab, certolizumab, golimumab or AME-527.

In one embodiment, an MRD-containing antibody binds IL1 alpha and IL1 beta. In another embodiment, an MRD-containing antibody binds IL1 beta and TNFSF11 (RANKL). In an additional embodiment, an MRD-containing antibody binds IL1 beta and a target selected from IL13, IL17A, TNF, VEGF, PGE2, VEGFR1, VEGFR2, TNFSF12 (TWEAK) and TNF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind IL1 beta and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds IL1 beta. In further embodiments, the antibody component of the MRD-containing antibody is catumaxomab, Xoma052, canakinumab or ACZ885. In additional embodiments, the antibody component of the MRD-containing antibody competes for IL1 alpha or IL1 beta binding with catumaxomab, Xoma052, canakinumab or ACZ885.

In another embodiment, an MRD-containing antibody binds IL12. In a further embodiment, an MRD-containing antibody binds IL12 and additionally binds IL18 or TNFSF12 (TWEAK). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CTLA-4and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CTLA-4. In further embodiments, the antibody component of the MRD-containing antibody is briakinumab or ustekinumab. In additional embodiments, the antibody component of the MRD-containing antibody competes for IL12 binding with briakinumab or ustekinumab.

In another embodiment, an MRD-containing antibody binds CTLA-4. In a further embodiment, an MRD-containing antibody binds CTLA4 and additionally binds PDL-1 or BTNO2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CTLA-4 and one or both of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CTLA-4. In further embodiments, the antibody component of the MRD-containing antibody is tremelimumab or iplimumab. In additional embodiments, the antibody component of the MRD-containing antibody competes for CTLA-4 binding with tremelimumab or iplimumab.

In an additional embodiment, an MRD-containing binds IL13. In a further embodiment, an MRD-containing antibody binds IL13 and additionally binds a target selected from: IL1beta, IL4, IL9, IL13, IL25, a LHR agonist, MDC, MIF, PED2, SPRR2a, SPRR2b; TARC, TGF-beta and IL25. In another embodiment, an MRD-containing antibody binds IL13 and a target selected from IL5, ADAM8, a LHR (agonist), IL23p19 and IgE. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind IL13 and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds IL13. In further embodiments, the antibody component of the MRD-containing antibody is TNX-650, lebrikizumab or CAT354. In additional embodiments, the antibody component of the MRD-containing antibody competes for IL13 binding with TNX-650, lebrikizumab or CAT354.

In a further embodiment, an MRD-containing antibody binds RGM A. In a further embodiment, an MRD-containing antibody binds RGM A and additionally binds a target selected from: RGM B, MAG, NgR, NogoA, OMGp and CSPGs. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind RGM A and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds RGM A.

In another embodiment, an MRD-containing antibody binds CD38 and additionally binds a target selected from CD20, TNFRSF5 (CD40) ALK1, TNF, VEGF, VEGFA, VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, VEGFR1, VEGFR2 and CD138. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD38 and at least 1, 2 or all 3 of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD38. In further embodiments, the antibody component of the MRD-containing antibody binds MOR202 or daratumumab. In additional embodiments, the antibody component of the MRD-containing antibody competes for CD38 binding with MOR202 or daratumumab.

In some embodiments an MRD-containing antibody binds ErbB1 (EGFR) and additionally binds ErbB3. In specific embodiments, the antibody component of the MRD-containing antibody binds ErbB1. In additional embodiments, the antibody component of the MRD-containing antibody is ERBITUX®. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB1-binding with ERBITUX®. In another embodiment, the antibody component of the MRD-containing antibody is an ErbB1-binding antibody selected from: nimotuzumab, zalutumumab, matuzumab, panitumumab, MEDX-214, and ABX-EGF. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB1-binding with an antibody selected from: nimotuzumab, zalutumumab, matuzumab, panitumumab, MEDX-214, and ABX-EGF.

In one embodiment, an MRD-containing antibody binds ErbB2 and IGF1R. In another embodiment, an MRD-containing antibody binds ErbB2, Ang2, and IGF1R. In specific embodiments, the antibody component of the MRD-containing antibody binds ErbB2. In additional embodiments, the antibody component of the MRD-containing antibody is HuMax-Her2™ or trastuzumab-DM1. In further embodiments, the antibody component of the MRD-containing antibody is trastuzumab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB2-binding with trastuzumab.

In one embodiment, an MRD-containing antibody binds ErbB2 and additionally binds a target selected from: ErbB3, EGFR, IGF1R, cMet, VEGF, RON (MST1R), DLL4, PLGF, CDCP1 (CD318), NRP1, TNFRSF10A (DR4) and TNFRSF10B (DR5). In another embodiment, an MRD-containing antibody binds ErbB2 and additionally binds a target selected from: CD2, CD3, CD4 and NKG2D. In an additional embodiment, an MRD-containing antibody binds ErbB2 and IGF1, IGF2 or IGF1,2. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind ErbB2 and additionally bind 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds ErbB2. In additional embodiments, the antibody component of the MRD-containing antibody is HuMax-Her2™ or trastuzumab-DM1. In further embodiments, the antibody component of the MRD-containing antibody is trastuzumab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB2-binding with trastuzumab.

In some embodiments an MRD-containing antibody binds ErbB2 and additionally binds ErbB3. In specific embodiments, the antibody component of the MRD-containing antibody binds ErbB2. In additional embodiments, the antibody component of the MRD-containing antibody is HuMax-Her2™ or trastuzumab-DM1. In further embodiments, the antibody component of the MRD-containing antibody is trastuzumab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB2-binding with trastuzumab. In another embodiment, the antibody component of the MRD-containing antibody is an ErbB2-binding antibody selected from: MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics), MGAH22 (MacroGenics), and pertuzumab (OMNITARG™). In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for ErbB2-binding with an antibody selected from: MDX-210, tgDCC-E1A, MGAH22, and pertuzumab.

In some embodiments, an MRD-containing antibody binds ErbB2 and HER2/3. In further embodiments, an MRD-containing antibody binds ErbB2 and HER2/3 simultaneously.

Angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathways have been observed to provide at best transitory therapeutic benefits followed by restoration of tumor growth and progression due to an apparent ability of angiogenic tumors to adapt the presence of these inhibitors. Without being bound by theory, it is believed that the multivalent and multispecific properties of MRD-containing antibodies that bind an angiogenesis target provide these compounds with an ability to extend anti-angiogenic therapeutic benefits beyond those observed from for example, conventional monoclonal antibody therapies by binding multiple distinct angiogenesis related targets and thereby disrupting resistance mechanisms available to the angiogenic tumor.

In another embodiment, an MRD-containing antibody binds PDGFRA and additionally binds an target selected from: VEGFA, VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, IGF1, IGF2, IGF1,2, HGF, TIE2, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, EGFR, cMET, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind PDGFRA and binds at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds PDGFRA. In further embodiments, the antibody component of the MRD-containing antibody is olaratumab. In further embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for PDGFRA binding with olaratumab. In further embodiments, the antibody component of the MRD-containing antibody is MEDI-575. In further embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for PDGFRA binding with MEDI-575.

In another embodiment, an MRD-containing antibody binds PDGFRB and additionally binds an target selected from: VEGFA, VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, IGF1, IGF2, IGF1,2, HGF, TIE2, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, EGFR, cMET, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, and PDGFRA. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind PDGFRB and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds PDGFRB.

In another embodiment, an MRD-containing antibody binds VEGFR1 and additionally binds an angiogenic target selected from: VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGFR1 and additionally bind 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGFR1. In further embodiments, the antibody component of the MRD-containing antibody is IMC-18F1. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for VEGFR1 binding with IMC-18F1.

In another embodiment, an MRD-containing antibody binds VEGFR2 and additionally binds a target selected from: VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, NRP1, ROBO4, CD30, CD33, CD55 CD80, KIT, CXCL12, Notch1EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, 1L18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR3, Notch 1, DLL4, EGFL7, α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind VEGFR2 and additionally bind 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGFR2. In further embodiments, the antibody component of the MRD-containing antibody is IMC-1C11 or DC101. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for VEGFR2 binding with IMC-1C11 or DC101.

In another embodiment, an MRD-containing antibody binds VEGFR2 and additionally binds ANG2 or TIE2. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGFR2. In further embodiments, the antibody component of the MRD-containing antibody is IMC-1C11, DC101 or TTAC-0001. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for VEGFR2 binding with IMC-1C11, DC101 or TTAC-0001. In further embodiments, the TIE2 binding component comprises a fragment of ANG2 that binds TIE2. In particular embodiments, the TIE2 binding component comprises amino acids 283-449 of the human ANG2 disclosed in NCBI Ref Seq. No. NP_001138.1.

In another embodiment, an MRD-containing antibody binds DLL4 and additionally binds a target selected from: EGFR, PLGF, VEGFR1, VEGFR2 and VEGF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind DLL4 and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In further embodiments, the antibody component of the MRD-containing antibody is REGN421. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for DLL4 binding with REGN421.

In additional embodiments, an MRD-containing antibody binds to an anti-angiogenic and a metastatic or invasive cancer target. In one embodiment, an MRD-containing antibody binds to an angiogenic target and also binds a metastatic or invasive cancer target selected from: CXCL12, CXCR4 (e.g., CXCR4b), CCR7 (e.g., CXCR7b), CD44 (e.g., CD44v3 and CD44v6), α2β1 integrin, α4β1 integrin, α5β1 integrin, αvβ1 integrin, αvβ3 integrin, TGFb, αvβ5 integrin, α9β1 integrin, α6β4 integrin, αMβ2 integrin, PD-1, HGF, cMET, MMP2, MMP-7, MMP-9, MMP-12, VEGFA, VEGFB, and IGF1. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind an angiogenic target and also bind 2, 3, 4, 5 or more of these metastatic or invasive cancer targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds VEGF. In further embodiments, the antibody component of the MRD-containing antibody is bevacizumab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for VEGF binding with bevacizumab.

In one embodiment, an MRD-containing antibody binds to 2 or more targets associated with distinct cell signaling pathways. In additional embodiments, an MRD-containing antibody binds to 2 or more targets associated with redundant, overlapping or cross-talking signaling pathways. For example, in one embodiment, an MRD-containing antibody binds to 2 or more targets associated with PI3K/AKT/mTOR signaling (e.g., ErbB2, EGFR, IGF1R, Notch, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, FGFR4, GPCR, and/or c-MET). In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with receptor tyrosine Raf/MEK/MAPK signaling (e.g., VEGFR1, VEGFR2, VEGFR3, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, FGFR4, CD28, RET, cMET, EGFR, ErbB2, Notch, Notch1, Notch3, Notch4, DLL1, DLL4, Jagged, Jagged1, Jagged2, and Jagged3. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 1, 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with SMAD signaling (e.g., Notch, TGFβ, TGFβR1, TGFβR2, and a BMP). In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with JAK/STAT signaling (e.g., IFNgR1, IFNgR3, IFNG, IFN-AR2, IFN-AR1, IFN alpha, IFN beta, IL6a receptor (GP130), IL6, IL12RB1, IL12, and EGFR). Thus, the invention encompasses an MRD-containing antibody that binds to 2 or more targets selected from WNT1, WNT2, WNT2b, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4, DLL1, DLL4, Jagged, Jagged1, Jagged2, and Jagged3. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with NFkB signaling (e.g., BCR, TCR, IL1R, ILL FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1 (TNFb, LTa), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF13 (APRIL), TNFSF13B (BLYS), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A), TNFRSF17 (BCMA), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), TNFRSF25 (DR3), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), TNFSF2 (TNFa), TNFSF3 (LTb), TNFRSF3 (LTBR), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM), TNFSF18 (GITR Ligand), TNFRSF18 (GITR), TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), a BMP, NGF, and TGF alpha). In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with cell proliferation (e.g., FGF1, FGF2, FGF7, FGF4, FGF10, FGF18b, FGF19, FGF23, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFRIIIB and FGFR-IIIC), FGFR3, FGFR4, TCR, TNFRSF5 (CD40), TLR1, TLR2, TLR3, TLR 4, TLR5, and TLR6). In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with toll-like receptor signaling (e.g., TLR1, TLR2, TLR3, TLR 4, TLR5, and TLR6).

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with B cell signaling (e.g., mIg, Igα/Igβ (CD79a/CD79b) heterodimers (α/β), CD19, CD20, CD21, CD22, CD23, CD27, CD30, CD46, CD80, CD86, ICOSL (B7-H2), HLA-DR (CD74), PD1, PDL1, TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), BTLA, TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), Fc gamma RIIB, IL4R and CRAC. In a particular embodiment, the MRD-containing antibody binds to CD19 and CD20. In an additional embodiment, the MRD-containing antibody binds CD19, CD20, and CD22. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) binds 2, 3, 4, 5 or more of these targets.

In a further embodiment, an MRD-containing antibody binds to 1 or more B cell surface markers selected from: CD10, CD24, CD37, CD53, CD72, CD75, CD77, CD79a, CD79b, CD81, CD82, CD83, CD84 (SLAM5) and CD85. In a further embodiment, an MRD-containing antibody binds to 1 or more B cell surface markers selected from: CD10, CD24, CD37, CD53, CD72, CD75, CD77, CD79a, CD79b, CD81, CD82, CD83, CD84 (SLAM5) and CD85. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these B cell surface markers.

In additional embodiments, an MRD-containing antibody binds CD19 and a target selected from: CD20, CD22, CD30, CD33, TNFRSF5 (CD40), CD52, CD74, CD80, CD138, VEGFR1, VEGFR2, EGFR, TNFRSF10A (DR4), TNFRSF10B (DR5), TNF, NGF, VEGF, IGF1,2, IGF2, IGF1 and TNFSF11 (RANKL). In additional embodiments, an MRD-containing antibody binds CD20 and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD19 and also bind at least 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD19. In further embodiments, the antibody component of the MRD-containing antibody is MDX-1342, SGN-CD19A, XMAB®5574, SGN-19A, ASG-5ME or MEDI-551. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for CD19 binding with MDX-1342, SGN-CD19A, XMAB®5574, SGN-19A, ASG-5ME or MEDI-551.

In additional embodiments, an MRD-containing antibody binds CD22 and a target selected from: CD19, CD20, CD23, CD30, CD33, TNFRSF5 (CD40), CD52, CD74, CD80, TNFRSF10A (DR4), TNFRSF10B (DR5), VEGF, TNF and NGF. In additional embodiments, an MRD-containing antibody binds CD22 and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD22 and also bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD22. In further embodiments, the antibody component of the MRD-containing antibody is epratuzumab or inotuzumab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for CD22 binding with epratuzumab or inotuzumab.

In additional embodiments, the antibody component of the MRD-containing antibody is moxetumomab (CAT-8015, Cambridge Antibody Technologies). In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for CD22 binding with moxetumomab.

In additional embodiments, an MRD-containing antibody binds TNFRSF5 (CD40) and a target selected from: BCMA, TNFSF11 (RANKL), VEGFR1, VEGFR2, TNFRSF10A (DR4), TNFRSF10B (DR5), CD22, CD30, CD38, CD56 (NCAM), CD70, CD80, CD138, IL6, IGF1, IGF2, IGF1,2, BLyS, APRIL and NGF. In additional embodiments, an MRD-containing antibody binds CD40 and a target selected from: CD3, CD4 and NKG2D. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD40 and also bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD40. In further embodiments, the antibody component of the MRD-containing antibody is CP870893, dacetuzumab, ANTOVA®, lucatumumab, XMAB®5485 or teneliximab. In additional embodiments, the antibody component, MRD component, and/or MRD-containing antibody competes for CD40 binding with CP870893, dacetuzumab, ANTOVA®, lucatumumab, XMAB®5485 or teneliximab.

In some embodiments, an MRD-containing antibody binds CD33 and a target selected from: FLT3, CD44, TNFRSF10A (DR4), TNFRSF10B (DR5), CD80, MGC, VEGFR1, VEGFR2, IL1, IL6, TNF and VEGF. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNFRSF10B and also bind at least 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD33. In further embodiments, the antibody component of the MRD-containing antibody is gemtuzumab or lintuzumab. In additional embodiments the antibody component, MRD component, and/or MRD-containing antibody competes for CD33 binding with gemtuzumab or lintuzumab.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with antigen presentation cell signaling (e.g., mIg, Igα/Igβ (CD79a/CD79b) heterodimers (α/β), CD19, CD20, CD21, CD22, CD23, CD27, CD28, CD30, CD30L, TNFSF14 (LIGHT, HVEM Ligand), CD70, ICOS, ICOSL (B7-H2), CTLA4, PD-1, PDL1 (B7-H1), B7-H4, B7-H3, PDL2 (B7-DC), BTLA, CD46, CD80 (B7-1), CD86 (B7-2), HLA-DR, CD74, PD1, TNFRSF4 (OX40), TNFRSF9 (41BB), TNFSF4 (OX40 Ligand), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), BTLA, TNFRSF18 (GITR), MHC-1, TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), Fcgamma RIIB, IL4R and CRAC). In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In another embodiment, an MRD-containing antibody binds to 2 or more targets associated with T cell receptor signaling (e.g., CD3, CD4, CD27, CD28, CD70, IL2R, LFA-1, C4, ICOS, CTLA-4, CD45, CD80, CD86, PG-1, TIM1, TIM2, TIM3, TIM4, galectin 9, TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF21 (DR6), TNFRSF6 (Fas, CD95), TNFRSF25 (DR3), TNFRSF14 (HVEM), TNFSF18, TNFRSF18 (GITR), TNFRSF4 (OX40), TNFSF4 (OX40 Ligand), PD1, PDL1, CTLA4, TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand), TNFSF5 (CD40 Ligand), BTLA, and CRAC). In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these targets.

In additional embodiments, an MRD-containing antibody binds to a cell surface tumor antigen and a second target that is associated with an escape pathway for resisting chemotherapy. In some embodiments, the cell surface tumor antigen is a member selected from EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC.

In further embodiments, an MRD-containing antibody binds P-glycoprotein (encoded by MDR1) and a cell surface antigen. In particular embodiments, the cell surface antigen is a tumor antigen. In further embodiments, the cell surface tumor antigen is a member selected from EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC. In particular embodiments the MRD-containing antibody competes for CD30 binding with SGN-35 (brentuximab). In additional particular embodiments the MRD-containing antibody competes for ErbB2 binding with trastuzumab.

In further embodiments, an MRD-containing antibody binds MRP (Multidrug-Resistance associated Protein) and a cell surface antigen. In particular embodiments, the cell surface antigen is a tumor antigen. In further embodiments, the cell surface tumor antigen is a member selected from EGFR, ErbB2, ErbB3, ErbB4, FGFR, VEGFR1, VEGFR2, VEGFR3, PDGFR1, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD56, CD70, CD133, CD138, FOLR1, IGF1-R, Cripto, SLC44A4 and GCC. In particular embodiments the MRD-containing antibody competes for CD30 binding with SGN-35 (brentuximab). In additional particular embodiments the MRD-containing antibody competes for ErbB2 binding with trastuzumab.

In another embodiments an MRD-containing antibody binds to a therapeutic target and a second target that is associated with an escape pathway for resisting the therapeutic effect resulting from targeting the therapeutic target. For example, in one embodiment, an MRD-containing antibody binds to EGFR and a target selected from MDR1, cMET, Notch, Notch1, Notch3, Notch4, DLL1, DLL4, Jagged, Jagged1, Jagged2, and Jagged3. In some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) binds 2, 3, 4, 5 or more of these targets.

In specific embodiments, the MRD-containing antibody targets ErbB2 and an angiogenic factor. In specific embodiments, the MRD-containing antibody targets ErbB2 and IGF1R. In another embodiment, the antibody targets ErbB2 and at least one MRD targets an angiogenic factor and/or IGF1R. In one embodiment, an antibody that binds to the same ErbB2 epitope as trastuzumab is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In an additional embodiment, an antibody that competitively inhibits trastuzumab binding is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In additional embodiments, an antibody that comprises the sequences of SEQ ID NOs:59-64 is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In additional embodiments, the trastuzumab antibody is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R.

In some embodiments, an antibody that binds to ErbB2 is operably linked to an MRD that targets Ang2. In some embodiments, the antibody that binds to ErbB2 is linked to an Ang2 binding MRD that binds to the same Ang2 epitope as an MRD comprising the sequence of MGAQTNFMPMDNDELLLYEQFILQQGLE SEQ ID NO:8. In some embodiments, the antibody that binds to ErbB2 is linked to an Ang2 binding MRD that competitively inhibits an MRD comprising the sequence of SEQ ID NO:8. In some embodiments, the antibody that binds to ErbB2 is linked to an MRD comprising the sequence of SEQ ID NO:8.

In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to ErbB2. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to ErbB2. In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to ErbB2. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to ErbB2.

In some embodiments, at least one Ang2 binding MRD is operably linked directly to an antibody that binds to ErbB2. In additional embodiments, at least one Ang2 binding MRD is operably linked to an antibody that binds to ErbB2 via a linker.

In some embodiments, an antibody that binds to ErbB2 is operably linked to an MRD that targets IGF1R. In some embodiments, the antibody that binds to ErbB2 is linked to an IGF1R binding MRD that binds to the same IGF1R epitope as an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds to ErbB2 is linked to an IGF1R binding MRD that competitively inhibits an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds to ErbB2 is linked to an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence SLFVPRPERK (SEQ ID NO:103). In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence ESDVLHFTST (SEQ ID NO:104). In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence LRKYADGTL (SEQ ID NO:105).

In some embodiments, at least one IGF1R binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to ErbB2. In some embodiments, at least one IGF1R binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to ErbB2. In some embodiments, at least one IGF1R binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to ErbB2. In some embodiments, at least one IGF1R binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to ErbB2.

In some embodiments, at least one IGF1R binding MRD is operably linked directly to an antibody that binds to ErbB2. In additional embodiments, at least one IGF1R binding MRD is operably linked to an antibody that binds to ErbB2 via a linker.

In some embodiments, an MRD-containing antibody targets ErbB2 and HER2/3. In some embodiments, an MRD-containing antibody can bind to ErbB2 and HER2/3 simultaneously. In some embodiments, an antibody that binds to ErbB2 is operably linked to an MRD that targets HER2/3. In additional embodiments, at least one HER2/3-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to ErbB2. In further embodiments, at least one HER2/3-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to ErbB2. In additional embodiments, at least one HER2/3-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to ErbB2. In additional embodiments, at least one HER2/3-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to ErbB2.

In some embodiments, at least one HER2/3-binding MRD is operably linked directly to an antibody that binds to ErbB2. In additional embodiments, at least one HER2/3-binding MRD is operably linked to an antibody that binds to ErbB2 via a linker.

In some embodiments, an MRD-containing antibody targets ErbB2 and HER2/3. In some embodiments, an MRD-containing antibody can bind to ErbB2 and HER2/3 simultaneously. In some embodiments, an antibody that binds to HER2/3 is operably linked to an MRD that targets ErbB2. In additional embodiments, at least one ErbB2-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to HER2/3. In further embodiments, at least one ErbB2-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to HER2/3. In additional embodiments, at least one ErbB2-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to HER2/3. In additional embodiments, at least one ErbB2-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to HER2/3.

In some embodiments, at least one ErbB2-binding MRD is operably linked directly to an antibody that binds to HER2/3. In additional embodiments, at least one ErbB2-binding MRD is operably linked to an antibody that binds to HER2/3 via a linker.

In some embodiments, the MRD-containing antibody targets ErbB2, Ang2, and IGF1R. In some embodiments, the MRD-containing antibody comprises an antibody that targets ErbB2, an MRD that targets Ang2, and an MRD that targets IGF1R. In some embodiments, the Ang2 and IGF1R MRDs are attached to the same location on the anti-ErbB2 antibody. In some embodiments, the Ang2 and IGF1R MRDs are attached to different locations on the anti-ErbB2 antibody. In some embodiments, the Ang2 and IGF1R MRDs are on the light chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 and IGF1R MRDs are on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the light chain of the ErbB2 antibody, and the IGF1R MRD is on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the heavy chain of the ErbB2 antibody, and the IGF1R MRD is on the light chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the IGF1R MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. In some embodiments, the IGF1R MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the Ang2 MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets Ang2, an MRD that targets ErbB2, and an MRD that targets IGF1R; and multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets IGF1R, an MRD that targets ErbB2, and an MRD that targets Ang2 are also encompassed by the invention.

In some embodiments, the MRD-containing antibody targets ErbB2, Ang2, and HER2/3. In some embodiments, the MRD-containing antibody comprises an antibody that targets ErbB2, an MRD that targets Ang2, and an MRD that targets HER2/3. In some embodiments, the Ang2 and HER2/3 MRDs are attached to the same location on the anti-ErbB2 antibody. In some embodiments, the Ang2 and HER2/3 MRDs are attached to different locations on the anti-ErbB2 antibody. In some embodiments, the Ang2 and HER2/3 MRDs are on the light chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 and HER2/3 MRDs are on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the light chain of the ErbB2 antibody, and the HER2/3 MRD is on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the heavy chain of the ErbB2 antibody, and the HER2/3 MRD is on the light chain of the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the HER2/3 MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3 MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the Ang2 MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets HER2/3, an MRD that targets ErbB2, and an MRD that targets Ang2; and multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets Ang2, an MRD that targets ErbB2, and an MRD that targets HER2/3 are also encompassed by the invention.

In some embodiments, the MRD-containing antibody targets ErbB2, HER2/3, and IGF1R. In some embodiments, the MRD-containing antibody comprises an antibody that targets ErbB2, an MRD that targets HER2/3, and an MRD that targets IGF1R. In some embodiments, the HER2/3 and IGF1R MRDs are attached to the same location on the anti-ErbB2 antibody. In some embodiments, the HER2/3 and IGF1R MRDs are attached to different locations on the anti-ErbB2 antibody. In some embodiments, the HER2/3 and IGF1R MRDs are on the light chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3 and IGF1R MRDs are on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3 MRD is on the light chain of the ErbB2 antibody, and the IGF1R MRD is on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3 MRD is on the heavy chain of the ErbB2 antibody, and the IGF1R MRD is on the light chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3 MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the IGF1R MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. In some embodiments, the IGF1R MRD is on the N-terminus of the heavy chain of the ErbB2 antibody, and the HER2/3 MRD is on the C-terminus of the light chain of the anti-ErbB2 antibody. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets HER2/3, an MRD that targets ErbB2, and an MRD that targets IGF1R; and multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets IGF1R, an MRD that targets ErbB2, and an MRD that targets HER2/3 are also encompassed by the invention.

In some embodiments, the MRD-containing antibody targets ErbB2, Ang2, HER2/3, and IGF1R. In some embodiments, the MRD-containing antibody comprises an antibody that targets ErbB2, an MRD that targets Ang2, an MRD that targets HER2/3, and an MRD that targets IGF1R. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are attached to the same chain of the anti-ErbB2 antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are attached to different chains of the anti-ErbB2 antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are on the light chain of the anti-ErbB2 antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are on the heavy chain of the anti-ErbB2 antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are attached to the same terminus of the anti-ErbB2 antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are attached to different termini of the anti-ErbB2 antibody. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising: an antibody that targets HER2/3, an MRD that targets ErbB2, an MRD that targets Ang2, and an MRD that targets IGF1R; multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets Ang2, an MRD that targets ErbB2, an MRD that targets HER2/3, and an MRD that targets IGF1R; and multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising an antibody that targets IGF1R, an MRD that targets ErbB2, an MRD that targets HER2/3, and an MRD that targets Ang2 are also encompassed by the invention.

In some embodiments, the anti-ErbB2 antibody operably linked to an Ang2 binding MRD binds to both ErbB2 and Ang2 simultaneously. In some embodiments, the anti-ErbB2 antibody operably linked to an IGF1R binding MRD binds to both ErbB2 and IGF1R simultaneously. In some embodiments, the anti-ErbB2 antibody operably linked to a HER2/3 binding MRD binds to both ErbB2 and HER2/3 simultaneously. In some embodiments, the anti-ErbB2 antibody operably linked to an Ang2 MRD, an IGF1R MRD, and/or a HER2/3 MRD binds to ErbB2, Ang2, IGF1R, and/or HER2/3 simultaneously. In some embodiments, the anti-ErbB2 antibody operably linked to an Ang2, IGF1R and/or HER2/3 binding MRD(s) exhibits ADCC activity. In additional embodiments, the anti-ErbB2 antibody operably linked to an Ang2, IGF1R, and/or HER2/3 binding MRD(s) down-regulates Akt signaling. In additional embodiments, the anti-ErbB2 antibody operably linked to an Ang2 binding MRD inhibits Ang2 binding to TIE2. In additional embodiments, the anti-ErbB2 antibody operably linked to an IGF1R binding MRD(s) down-regulates IGF1R signaling. In additional embodiments, the anti-ErbB2 antibody operably linked to an Ang2, IGF1R and/or HER2/3 binding MRD(s) inhibits cell proliferation. In additional embodiments, the anti-ErbB2 antibody operably linked to an Ang2, IGF1R, and/or HER2/3 binding MRD(s) inhibits tumor growth.

In specific embodiments, the MRD-containing antibody targets VEGF and an angiogenic factor. In specific embodiments, the MRD-containing antibody targets VEGF and IGF1R. In another embodiment, the antibody targets VEGF and at least one MRD targets an angiogenic factor and/or IGF1R. In one embodiment, an antibody that binds to the same VEGF epitope as bevacizumab is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In an additional embodiment, an antibody that competitively inhibits bevacizumab binding is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In additional embodiments, an antibody that comprises the sequences of SEQ ID NOs:78-79 is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R. In additional embodiments, the bevacizumab antibody is operably linked to at least one MRD that targets an angiogenic factor and/or IGF1R.

In some embodiments, an antibody that binds to VEGF is operably linked to an MRD that targets Ang2. In some embodiments, the antibody that binds to VEGF is linked to an Ang2 binding MRD that binds to the same Ang2 epitope as an MRD comprising the sequence of SEQ ID NO:8. In some embodiments, the antibody that binds to VEGF is linked to an Ang2 binding MRD that competitively inhibits an MRD comprising the sequence of SEQ ID NO:8. In some embodiments, the antibody that binds to VEGF is linked to an MRD comprising the sequence of SEQ ID NO:8.

In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to VEGF. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to VEGF. In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to VEGF. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to VEGF.

In some embodiments, at least one Ang2 binding MRD is operably linked directly to an antibody that binds to VEGF. In additional embodiments, at least one Ang2 binding MRD is operably linked to an antibody that binds to VEGF via a linker.

In some embodiments, an antibody that binds to VEGF is operably linked to an MRD that targets IGF1R. In some embodiments, the antibody that binds to VEGF is linked to an IGF binding MRD that binds to the same IGF epitope as an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds to VEGF is linked to an IGF1R binding MRD that competitively inhibits an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds to VEGF is linked to an MRD comprising the sequence of SEQ ID NO:14. In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence SLFVPRPERK (SEQ ID NO:103). In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence ESDVLHFTST (SEQ ID NO:104). In some embodiments, the antibody that binds ErbB2 is linked to an MRD encoding the sequence LRKYADGTL (SEQ ID NO:105).

In some embodiments, at least one IGF1R binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to VEGF. In some embodiments, at least one IGF1R binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to VEGF. In some embodiments, at least one IGF1R binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to VEGF. In some embodiments, at least one IGF1R binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to VEGF.

In some embodiments, at least one IGF1R binding MRD is operably linked directly to an antibody that binds to VEGF. In additional embodiments, at least one IGF1R binding MRD is operably linked to an antibody that binds to VEGF via a linker.

In some embodiments, the MRD-containing antibody targets VEGF, Ang2, and IGF1R. In some embodiments, the MRD-containing antibody comprises an antibody that targets VEGF, an MRD that targets Ang2, and an MRD that targets IGF1R. In some embodiments, the Ang2 and IGF1R MRDs are attached to the same location on the anti-VEGF antibody. In some embodiments, the Ang2 and IGF1R MRDs are attached to different locations on the anti-VEGF antibody. In some embodiments, the Ang2 and IGF1R MRDs are on the light chain of the anti-VEGF antibody. In some embodiments, the Ang2 and IGF1R MRDs are on the heavy chain of the anti-VEGF antibody. In some embodiments, the Ang2 MRD is on the light chain of the anti-VEGF antibody, and the IGF1R MRD is on the heavy chain of the anti-VEGF antibody. In some embodiments, the Ang2 MRD is on the heavy chain of the anti-VEGF antibody, and the IGF1R MRD is on the light chain of the anti-VEGF antibody. In some embodiments, the Ang2 MRD is on the N-terminus of the heavy chain of the anti-VEGF antibody, and the IGF1R MRD is on the C-terminus of the light chain of the anti-VEGF antibody. In some embodiments, the IGF1R MRD is on the N-terminus of the heavy chain of the anti-VEGF antibody, and the Ang2 MRD is on the C-terminus of the light chain of the anti-VEGF antibody.

In some embodiments, the anti-VEGF antibody operably linked to an Ang2 binding MRD binds to both anti-VEGF and Ang2 simultaneously. In some embodiments, the anti-VEGF antibody operably linked to an IGF1R binding MRD binds to both anti-VEGF and IGFR1 simultaneously. In some embodiments, the anti-VEGF antibody operably linked to an Ang2 binding MRD and an IGF1R binding MRD binds to VEGF, Ang2, and IGF1R simultaneously. In some embodiments, the anti-VEGF antibody operably linked to an Ang2 and/or IGF1R binding MRD(s) exhibits ADCC activity. In additional embodiments, the anti-VEGF antibody operably linked to an Ang2 and/or IGF1R binding MRD(s) down-regulates VEGF signaling. In additional embodiments, the anti-VEGF antibody operably linked to an Ang2 binding MRD inhibits Ang2 binding to TIE2. In additional embodiments, the anti-VEGF antibody operably linked to an IGF1R binding MRD inhibits IGF1R signaling. In additional embodiments, the anti-VEGF antibody operably linked to an Ang2 and/or IGF1R binding MRD(s) inhibits cell proliferation. In additional embodiments, the anti-VEGF antibody operably linked to an Ang2 and/or IGF binding MRD(s) inhibits tumor growth.

In some embodiments, the anti-ErbB2 antibody or the VEGF antibody contains and MRD that inhibits the binding of pertuzumab to ErbB2. In some embodiments, an anti-ErbB2 antibody contains at least one MRD that binds to Ang2 or IGF1R and one MRD that inhibits the binding of pertuzumab to ErbB2. In some embodiments, an anti-VEGF antibody contains at least one MRD that binds to Ang2 or IGF1R and one MRD that inhibits the binding of pertuzumab to ErbB2. In some embodiments, an anti-ErbB2 antibody contains an MRD that binds Ang2, an MRD that binds IGF1R, and an MRD that inhibits the binding of pertuzumab to ErbB2. In some embodiments, an anti-VEGF antibody contains an MRD that binds Ang2, an MRD that binds IGF1R, and an MRD that inhibits the binding of pertuzumab to ErbB2.

In specific embodiments, the MRD-containing antibody targets TNF and an angiogenic factor. In another embodiment, the antibody targets TNF, and at least one MRD targets an angiogenic factor. In one embodiment, an antibody that binds to the same TNF epitope as adalimumab is operably linked to at least one MRD that targets an angiogenic factor. In an additional embodiment, an antibody that competitively inhibits adalimumab binding is operably linked to at least one MRD that targets an angiogenic factor. In additional embodiments, an antibody that comprises the sequences of SEQ ID NOs:80-85 is operably linked to at least one MRD that targets an angiogenic factor. In additional embodiments, the adalimumab antibody is operably linked to at least one MRD that targets an angiogenic factor. In one embodiment, an antibody that binds to the same TNF epitope as golimumab is operably linked to at least one MRD that targets an angiogenic factor. In an additional embodiment, an antibody that competitively inhibits golimumab binding is operably linked to at least one MRD that targets an angiogenic factor. In additional embodiments, the golimumab antibody is operably linked to at least one MRD that targets an angiogenic factor.

In some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets Ang2. In some embodiments, the antibody that binds to TNF is linked to an Ang2 binding MRD that binds to the same Ang2 epitope as an MRD comprising the sequence of SEQ ID NO:8. In some embodiments, the antibody that binds to TNF is linked to an Ang2 binding MRD that competitively inhibits an MRD comprising the sequence of SEQ ID NO:8. In some embodiments, the antibody that binds to TNF is linked to an MRD comprising the sequence of SEQ ID NO:8.

In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds to TNF. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to TNF. In some embodiments, at least one Ang2 binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to TNF. In some embodiments, at least one Ang2 binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to TNF.

In some embodiments, at least one Ang2 binding MRD is operably linked directly to an antibody that binds to TNF. In additional embodiments, at least one Ang2 binding MRD is operably linked to an antibody that binds to TNF via a linker.

In some embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD binds to both TNF and Ang2 simultaneously. In some embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD exhibits ADCC activity. In additional embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD inhibits binding of TNF to the p55 and p75 cell surface TNF receptors. In additional embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD lyses surface TNF-expressing cells in vitro in the presence of complement. In additional embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD inhibits Ang2 binding to TIE2. In additional embodiments, the anti-TNF antibody operably linked to an Ang2 binding MRD reduces the signs and symptoms of arthritis.

In some embodiments, the MRD-containing antibody targets TNF and IL6. In some embodiments, the MRD-containing antibody is capable of binding TNF and IL6 simultaneously. Thus, in some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets IL6. In other embodiments, an antibody that binds to IL6 is operably linked to an MRD that targets TNF.

In some embodiments, at least one IL6-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds TNF. In some embodiments, at least one IL6-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to TNF. In some embodiments, at least one IL6-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to TNF. In some embodiments, at least one IL6-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to TNF.

In some embodiments, at least one TNF-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds IL6. In some embodiments, at least one TNF-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to IL6. In some embodiments, at least one TNF-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to IL6. In some embodiments, at least one TNF-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to IL6.

In some embodiments, at least one IL6-binding MRD is operably linked directly to an antibody that binds to TNF. In additional embodiments, at least one IL6-binding MRD is operably linked to an antibody that binds to TNF via a linker.

In some embodiments, at least one TNF-binding MRD is operably linked directly to an antibody that binds to IL6. In additional embodiments, at least one TNF-binding MRD is operably linked to an antibody that binds to IL6 via a linker.

In some embodiments, the MRD-containing antibody targets TNF and BLyS. In some embodiments, the MRD-containing antibody is capable of binding TNF and BLyS simultaneously. In some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets BLyS. In other embodiments, an antibody that binds to BLyS is operably linked to an MRD that targets TNF.

In some embodiments, at least one BLyS-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds TNF. In some embodiments, at least one BLyS-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to TNF. In some embodiments, at least one BLyS-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to TNF. In some embodiments, at least one BLyS-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to TNF.

In some embodiments, at least one TNF-binding MRD is operably linked to the C-terminus of the heavy chain of an antibody that binds BLyS. In some embodiments, at least one TNF-binding MRD is operably linked to the N-terminus of the heavy chain of an antibody that binds to BLyS. In some embodiments, at least one TNF-binding MRD is operably linked to the C-terminus of the light chain of an antibody that binds to BLyS. In some embodiments, at least one TNF-binding MRD is operably linked to the N-terminus of the light chain of an antibody that binds to BLyS.

In some embodiments, at least one BLyS-binding MRD is operably linked directly to an antibody that binds to TNF. In additional embodiments, at least one BLyS-binding MRD is operably linked to an antibody that binds to TNF via a linker.

In other embodiments, at least one TNF-binding MRD is operably linked directly to an antibody that binds to BLyS. In additional embodiments, at least one TNF-binding MRD is operably linked to an antibody that binds to BLyS via a linker.

In some embodiments, the MRD-containing antibody targets Ang2, TNF, and IL6. In some embodiments, the MRD-containing antibody is capable of binding Ang2, TNF, and IL6 simultaneously. In some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets Ang2 and an MRD that targets IL6. In some embodiments, the Ang2 and IL6-binding MRDs are located on the same antibody chain. In some embodiments, the Ang2 and IL6-binding MRDs are located on the same antibody terminus. In some embodiments, the Ang2 and IL6-binding MRDs are located on different antibody chains. In some embodiments, the Ang2 and IL6-binding MRDs are located on different antibody termini.

In some embodiments, an antibody that binds to Ang2 is operably linked to an MRD that targets TNF and an MRD that targets IL6. In some embodiments, the TNF and IL6-binding MRDs are located on the same antibody chain. In some embodiments, the TNF and IL6-binding MRDs are located on the same antibody terminus. In some embodiments, the TNF and IL6-binding MRDs are located on different antibody chains. In some embodiments, the TNF and IL6-binding MRDs are located on different antibody termini.

In some embodiments, an antibody that binds to IL6 is operably linked to an MRD that targets Ang2 and an MRD that targets TNF. In some embodiments, the Ang2 and TNF-binding MRDs are located on the same antibody chain. In some embodiments, the Ang2 and TNF-binding MRDs are located on the same antibody terminus. In some embodiments, the Ang2 and TNF-binding MRDs are located on different antibody chains. In some embodiments, the Ang2 and TNF-binding MRDs are located on different antibody termini.

In some embodiments, the MRD-containing antibody targets Ang2, TNF, and BLyS. In some embodiments, the MRD-containing antibody is capable of binding Ang2, TNF, and BLyS simultaneously. In some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets Ang2 and an MRD that targets BLyS. In other embodiments, an antibody that binds to BLyS is operably linked to an MRD that targets TNF and an MRD that targets Ang2. In other embodiments, an antibody that binds to Ang2 is operably linked to an MRD that targets TNF and an MRD that targets BLyS. In some embodiments, the Ang2, BLyS, and/or TNF-binding MRDs are located on the same antibody chain. In some embodiments, Ang2, BLyS, and/or TNF-binding MRDs are located on the same antibody terminus. In some embodiments, the Ang2, BLyS, and/or TNF-binding MRDs are located on different antibody chains. In some embodiments, the Ang2, BLyS, and/or TNF-binding MRDs are located on different antibody termini.

In some embodiments, the MRD-containing antibody targets Ang2, TNF, IL6, and BLyS. In some embodiments, the MRD-containing antibody is capable of binding Ang2, TNF, IL6 and BLyS simultaneously. In some embodiments, an antibody that binds to TNF is operably linked to an MRD that targets Ang2, an MRD that targets IL6, and an MRD that targets BLyS. In some embodiments, an antibody that binds to Ang2 is operably linked to an MRD that targets TNF, an MRD that targets IL6, and an MRD that targets BLyS. In some embodiments, an antibody that binds to IL6 is operably linked to an MRD that targets Ang2, an MRD that targets TNF, and an MRD that targets BLyS. In some embodiments, an antibody that binds to BLyS is operably linked to an MRD that targets Ang2, an MRD that targets IL6, and an MRD that targets TNF. In some embodiments, the TNF, Ang2, IL6, and/or BLyS-binding MRDs are located on the same antibody chain. In some embodiments, the TNF, Ang2, IL6 and/or BLyS-binding MRDs are located on the same antibody terminus. In some embodiments, the TNF, Ang2, IL6, and/or BLyS-binding MRDs are located on different antibody chains. In some embodiments, the TNF, Ang2, IL6 and/or BLyS-binding MRDs are located on different antibody termini.

VI. Methods of Making Antibody-MRD Fusions

The multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies) and MRDs can be produced by any method known in the art for the synthesis of antibodies, polypeptides, immunoconjugates, and cytotoxins, in particular, by chemical synthesis or by recombinant expression techniques. An advantage of multivalent and multispecific compositions (e.g., MRD-containing antibodies) is that they can be produced using protocols that are known in the art for producing antibodies. The antibody-MRD fusion molecules can be encoded by a polynucleotide comprising a nucleotide sequence. Thus, the polynucleotides described herein can encode an MRD, an antibody heavy chain, an antibody light chain, a fusion protein comprising an antibody heavy chain and at least one MRD, and/or a fusion protein comprising an antibody light chain and at least one MRD.

Accordingly, the invention provides vector constructs comprising a polynucleotide sequence(s) encoding multivalent and multispecific compositions (e.g., MRD-containing antibodies) and a host cell comprising these vector constructs. Standard techniques for cloning and transformation may be used in the preparation of cell lines expressing the multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention.

Recombinant expression vectors containing a polynucleotide sequence(s) encoding multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention can be prepared using well known techniques. The expression vectors include a polynucleotide coding sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences such as, those derived from mammalian, microbial, viral, or insect genes. Exemplary regulatory sequences present in the expression vector constructs include transcriptional promoters, operators, enhancers, mRNA ribosomal binding sites, and/or other appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleotide sequence for the appropriate polypeptide. Thus, a promoter sequence is operably linked to, for example, an antibody heavy chain-MRD sequence if the promoter nucleotide sequence controls the transcription of the appropriate nucleotide sequence.

The polynucleotide coding sequence in the expression vector can include additional heterologous sequences encoding polypeptides such as, signal peptides that are not naturally associated with antibody heavy and/or light chain sequences. For example, a nucleotide sequence for a signal peptide (secretory leader) can be fused in-frame to the polypeptide sequence so that the MRD-containing antibody is secreted to the periplasmic space or into the medium. A signal peptide that is functional in the intended host cells enhances extracellular secretion of the appropriate antibody. The signal peptide can be cleaved from the polypeptide upon secretion of antibody from the cell. Examples of sequences encoding secretory signals that can be included in the expression vectors include those described in for example, U.S. Pat. Nos. 5,698,435, 5,698,417, and 6,204,023.

A variety of host-expression vector systems can be utilized to express the coding sequence an MRD-containing antibody.

Host cells useful in the present invention include but are not limited to microorganisms such as, bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., Baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing MRD-containing antibody coding sequences. In particular embodiments, the mammalian cell systems are used to produce the multivalent and multispecific compositions of the invention (e.g., MRD-containing antibodies). Mammalian cell systems typically utilize recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Examples of mammalian host cells useful for producing the multivalent and multispecific compositions of the invention include, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, COS cells, 293 cells, 3T3 cells and hybridoma cells.

Vectors containing the polynucleotides encoding the multivalent and multispecific compositions of the invention (e.g., MRD containing antibodies) or portions or fragments thereof, include plasmid vectors, a single and double-stranded phage vectors, as well as single and double-stranded RNA or DNA viral vectors. The vectors can be routinely introduced into host cells using known techniques for introducing DNA and RNA into cells. Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated virus using known techniques for infection and transduction. Moreover, viral vectors may be replication competent or alternatively, replication defective. Alternatively, cell-free translation systems may also be used to produce the protein using RNAs derived from the DNA expression constructs of the invention (see, e.g., Intl. Appl. Publ. WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464).

Also provided herein, are methods of producing an MRD-containing antibody, the method comprising: culturing a host cell comprising one or more polynucleotides or an expression vector comprising one or more isolated polynucleotides in a medium under conditions allowing the expression of said one or more polynucleotide, wherein said one or more polynucleotides encodes one or more polypeptides that form part of MRD-containing antibody; and recovering said MRD-containing antibody.

Prokaryotes useful as host cells in producing the compositions of the invention (e.g., MRDs) include gram negative or gram positive organisms such as, E. coli and B. subtilis. Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or that supply an autotrophic requirement). Examples of useful prokaryotic host expression vectors include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA) and pRSET (Invitrogen, Calif., USA) series of vectors (see, e.g., Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7, (Rosenberg et al., Gene 56: 125-135 (1987)), beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978)); and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In alternative embodiments, eukaryotic host cell systems can be used, including yeast cells transformed with recombinant yeast expression vectors containing the coding sequence of an MRD-containing antibody of the present invention, such as, the expression systems taught in U.S. Pat. Appl. No. 60/344,169 and WO03/056914 (methods for producing human-like glycoprotein in a non-human eukaryotic host cell) (the contents of each of which are incorporated by reference in their entirety). Exemplary yeast that can be used to produce compositions of the invention, such as, MRDs, include yeast from the genus Saccharomyces, Pichia, Actinomycetes and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from a 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include, promoters from metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, (1980)) and other glycolytic enzymes, such as, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Additional suitable vectors and promoters for use in yeast expression as well as yeast transformation protocols are known in the art. See, e.g., Fleer et al., Gene, 107:285-195 (1991) and Hinnen et al., Proc. Natl. Acad. Sci., 75:1929 (1978).

Insect and plant host cell culture systems are also useful for producing the compositions of the invention. Such host cell systems include for example, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence of an MRD-containing antibody; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence of an MRD-containing antibody, including, but not limited to, the expression systems taught in U.S. Pat. No. 6,815,184, WO2004/057002, WO2004/024927, U.S. Pat. Appl. Nos. 60/365,769, 60/368,047, and WO2003/078614, the contents of each of which is herein incorporated by reference in its entirety.

In alternate embodiments, other eukaryotic host cell systems may be used, including animal cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus) including cell lines engineered to contain multiple copies of the DNA encoding an MRD-containing antibody either stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g., murine cell lines). In one embodiment, the vector comprising the polynucleotide(s) encoding the MRD-containing antibody of the invention is polycistronic.

Exemplary mammalian cells useful for producing these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells or hybridoma cells, other mammalian cells. Additional exemplary mammalian host cells that are useful in practicing the invention include but are not limited, to VERY, Hela, COS, MDCK, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. Some examples of expression systems and selection methods are described in the following references and references cited therein: Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000-2001), in Werner et al., Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), in Andersen and Krummen, Curr. Op. Biotechnol. 13:117-123 (2002), in Chadd and Chamow, Curr. Op. Biotechnol. 12:188-194 (2001), and in Giddings, Curr. Op. Biotechnol. 12: 450-454 (2001). Additional examples of expression systems and selection methods are described in Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984), Bittner et al., Methods in Enzymol. 153:51-544 (1987)). Transcriptional and translational control sequences for mammalian host cell expression vectors are frequently derived from viral genomes. Commonly used promoter sequences and enhancer sequences in mammalian expression vectors include, sequences derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus (CMV). Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen®) and pcDNA3 (Invitrogen®).

A number of selection systems can be used in mammalian host-vector expression systems, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes, which can be employed in tk, hgprt⁻ or aprt⁻ cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for e.g., dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase), and the glutamine synthase system.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an MRD-containing antibody along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1989).

A variety of host-expression vector systems may be utilized to express the coding sequence an MRD-containing antibody. A host cell strain can be chosen which modulates the expression of inserted antibody sequences, or modifies and processes the antibody gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

Stable expression typically achieves more reproducible results than transient expression and also is more amenable to large-scale production; however, it is within the skill of one in the art to determine whether transient expression is better for a particular situation. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the respective coding nucleic acids controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows selection of cells which have stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.

In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are expressed at levels (titers) comparable to those of antibodies. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are expressed at least about 10 μg/ml, at least about 20 μg/ml, or at least about 30 μg/ml. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are expressed at least about 40 μg/ml or at least about 50 μg/ml. In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are expressed at least about 60 μg/ml, at least about 70 μg/ml, at least about 80 μg/ml, at least about 90 μg/ml, at least about 95 μg/ml, at least about 100 μg/ml, at least about 110 μg/ml, at least about 120 μg/ml, at least about 130 μg/ml, at least about 140 μg/ml, at least about 150 μg/ml, at least about 160 μg/ml, at least about 170 μg/ml, at least about 180 μg/ml, at least about 190 μg/ml, or at least about 200 μg/ml. The expression levels of an antibody molecule can be increased by vector amplification and the use recombinant methods and tools known in the art, including chromatin remodeling strategies to enhance transgene expression.

The present invention is further directed to a method for modifying the glycosylation profile of an MRD-containing antibody that is produced by a host cell, comprising expressing in said host cell a nucleic acid encoding an MRD-containing antibody and a nucleic acid encoding a polypeptide with a glycosyltransferase activity, or a vector comprising such nucleic acids. Genes with glycosyltransferase activity include β(1,4)-N-acetylglucosaminyltransferase III (GnTII), α-mannosidase II (ManII), β(1,4)-galactosyltransferase (GalT), β(1,2)-N-acetylglucos aminyltransferase I (GnTI), and β(1,2)-N-acetylglucosaminyltransferase II (GnTII). In one embodiment, a combination of genes with glycosyltransferase activity are expressed in the host cell (e.g., GnTIII and Man II). Likewise, the method also encompasses expression of one or more polynucleotide(s) encoding the MRD-containing antibody in a host cell in which a glycosyltransferase gene has been disrupted or otherwise deactivated (e.g., a host cell in which the activity of the gene encoding α1-6 core fucosyltransferase has been knocked out). In another embodiment, the MRD-containing antibody can be produced in a host cell that further expresses a polynucleotide encoding a polypeptide having GnTIII activity to modify the glycosylation pattern. In a specific embodiment, the polypeptide having GnTIII activity is a fusion polypeptide comprising the Golgi localization domain of a Golgi resident polypeptide. In another embodiment, the expression of the MRD-containing antibody in a host cell that expresses a polynucleotide encoding a polypeptide having GnTIII activity results in an MRD-containing antibody with increased Fc receptor binding affinity and increased effector function. Accordingly, in one embodiment, the present invention is directed to a host cell comprising (a) an isolated nucleic acid comprising a sequence encoding a polypeptide having GnTIII activity; and (b) an isolated polynucleotide encoding an MRD-containing antibody of the present invention, such as, a chimeric, primatized or humanized antibody. In another embodiment, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain is the localization domain of mannosidase II. Methods for generating such fusion polypeptides and using them to produce antibodies with increased effector functions are disclosed in U.S. Provisional Pat. Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No. 2004/0241817, each of which is herein incorporated by reference.

The multivalent and multispecific compositions (e.g., MRD-containing antibodies) with altered glycosylation produced by the host cells of the invention typically exhibit increased Fc receptor binding affinity and/or increased effector function as a result of the modification of the host cell (e.g., by expression of a glycosyltransferase gene). The increased Fc receptor binding affinity can be increased binding to an Fcγ activating receptor, such as, the FcγRIIIa receptor. The increased effector function can be an increase in one or more of the following: increased antibody-dependent cellular cytotoxicity, increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased immune-complex-mediated antigen uptake by antigen-presenting cells, increased Fc-mediated cellular cytotoxicity, increased binding to NK cells, increased binding to macrophages, increased binding to polymorphonuclear cells (PMNs), increased binding to monocytes, increased crosslinking of target-bound antibodies, increased direct signaling inducing apoptosis, increased dendritic cell maturation, and increased T cell priming.

Once a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) of the invention has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In additional embodiments, the multivalent and multispecific compositions of the present invention or fragments thereof are optionally fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. In additional embodiments, the multivalent and multispecific compositions or fragments thereof are optionally fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. More particularly, it is envisioned that ligands (e.g., antibodies and other affinity matrices) for MRDs or other components of the multivalent and multispecific compositions can be used in affinity columns for affinity purification and that optionally, the MRDs or other components of the multivalent and monovalent multispecific composition that are bound by these ligands are removed from the composition prior to final preparation of the multivalent and multispecific compositions using techniques known in the art.

VII. Uses of Antibody-MRD Fusions

The multivalent and multispecific compositions (e.g., MRD-containing antibodies) described herein are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as, the treatment of cancer. In certain embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are useful for inhibiting tumor growth, reducing neovascularization, reducing angiogenesis, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vitro, ex vivo, or in vivo methods. Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the

Physician's Desk Reference (PDR). The PDR discloses dosages of the agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. The contents of the PDR are expressly incorporated herein in its entirety by reference. The 2006 edition of the Physician's Desk Reference (PDR) discloses the mechanism of action and preferred doses of treatment and dosing schedules for thalidomide (p 979-983), VELCADE® (p 2102-2106) and melphalan (p 976-979).

The multivalent and multispecific compositions (e.g., MRD-containing antibodies) are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The dosage ranges for the administration of the multivalent and multispecific compositions of the invention are those large enough to produce the desired effect in which the disease symptoms mediated by the target molecule are ameliorated. The dosage should not be so large as to cause adverse side effects, such as, hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or nonaqueous. However, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. Thus, an antibody-MRD containing composition can take the form of solutions, suspensions, tablets, capsules, sustained release formulations or powders, or other compositional forms.

In some embodiments, the compositions of the invention (e.g., multivalent and multispecific compositions (e.g., MRD-containing antibodies)) are formulated to ensure or optimize distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds and if so desired, the compositions are prepared so as to increase transfer across the BBB, by for example, formulation in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade Clin. Pharmacol. 29:685 (1989)).

The multivalent and multispecific binding properties of the compositions of the invention provide for the rational design of therapeutics preferentially directed to particular anatomical sites of interest in vivo. For example, in some embodiments, the MRD-containing antibody localizes to a site of inflammation or to a tumor site by binding of the antibody combining site to a molecule characterized by increased expression in the inflammation or tumor site, respectively. In particular embodiments, the binding affinity of the antibody combining site in an MRD-containing antibody for the target in vivo is at least 3×, at least 10×, at least 30× or at least 100× that of one or more MRD targets bound by the MRD-containing antibody (e.g., a surface antigen that is not otherwise considered a viable antibody target in view of for example, non-specific surface expression, or low target binding affinity associated with conventional antibody technologies).

Therapeutic formulations of the invention optionally contain pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 18th edition, Mack Publishing Co., Easton, Pa. 18042 [1990]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite, preservatives, isotonicifiers, stabilizers, metal complexes (e.g., Zn-protein complexes), and/or chelating agents such as EDTA. Physiologically tolerable carriers are well known in the art. Likewise, multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. Additional excipients useful in formulation the compositions of the invention include an agent which can act as: (1) a bulking agent, (2) a solubility enhancer, and (3) a stabilizer. Such excipients include, but are not limited to: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. In some embodiments buffers are present at a concentrations ranging from about 1 mM to about 200 mM, about 1 mM to about 100 mM or about 1 mM to about 50 mM. Suitable buffering agents for use in the formulations of the invention include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such, as Tris.

Stabilizers, referred to as tonicity modifiers, are present to adjust or maintain the tonicity of a liquid composition. When used with large, charged biomolecules such as proteins and antibodies, stabilizers interact with the charged groups of the amino acid side chains, to reduce the potential for inter and intra-molecular interactions. Examples of tonicity agents useful in preparing the formulations of the invention include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as, cottonseed oil, organic esters such as, ethyl oleate, and water-oil emulsions.

In one embodiment, a therapeutic composition contains a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) in an amount of at least 0.1 weight percent of MRD-containing antibody fusion per weight of total therapeutic composition. A weight percent is a ratio by weight of MRD-containing antibody per total composition. Thus, for example, 0.1 weight percent is 0.1 grams of MRD-containing antibody per 100 grams of total composition.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectable typically use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).

Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In one embodiment, the pharmaceutical composition formulations of the invention comprise a compound that is capable of reducing the viscosity of an aqueous formulation comprising multivalent and monovalent multispecific compositions. In one embodiment, the multivalent and monovalent multispecific composition is an MRD-containing antibody. In a further embodiment, the compound that is capable of reducing the viscosity of the formulation is selected from the group consisting of: methionine, arginine (e.g., arginine succinate), arginine dipeptide, arginine tripeptide, polyarginine, homoarginine, N-hydroxy-L-nor-arginine, nitroarginine methyl ester, argininamide, arginine methyl ester, arginine ethyl ester, lysine, lysinamide, lysine methyl ester, histidine, histidine methyl ester, histamine, alanine, alaninamide, alanine methyl ester, 2-amino-3-guanidino-propionic acid, guanidine, ornithine, agmatine, guanidobutyric acid, citrulline, putrescine, cadaverine, spermidine, spermine and urea. In some embodiments, the compound is present in the formulation at a concentration of at least 10 mM, at least 20 mM, at least 50 mM or at least 100 mM. In additional embodiments, the compound is present in the formulation at a concentration of between about 10 mM and 1 M. In further embodiments, the total protein concentration present in the formulation is at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml or at least 250 mg/ml.

The dosage schedule and amounts effective for therapeutic and prophylactic uses, i.e., the “dosing regimen”, will depend upon a variety of factors, including the cause, stage and severity of the disease or disorder, the health, physical status, age of the mammal being treated, and the site and mode of the delivery of the MRD-containing antibody. Therapeutic efficacy and toxicity of the complex and formation can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals. Data obtained from these procedures can likewise be used in formulating a range of dosages for human use. Moreover, therapeutic index (i.e., the dose therapeutically effective in 50 percent of the population divided by the dose lethal to 50 percent of the population (ED₅₀/LD₅₀)) can readily be determined using known procedures. The dosage is preferably within a range of concentrations that includes the ED₅₀ with little or no toxicity, and may vary within this range depending on the dosage form employed, sensitivity of the patient, and the route of administration.

The dosage regimen also takes into consideration pharmacokinetics parameters known in the art, such as, drug absorption rate, bioavailability, metabolism and clearance (see, e.g., Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611-617 (1996); Groning et al., Pharmazie 51:337-341 (1996); Fotherby Contraception 54:59-69 (1996); and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)). It is well within the state of the art for the clinician to determine the dosage regimen for each subject being treated. Moreover, single or multiple administrations of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) containing compositions can be administered depending on the dosage and frequency as required and tolerated by the subject. The duration of prophylactic and therapeutic treatment will vary depending on the particular disease or condition being treated. Some diseases are amenable to acute treatment whereas others require long-term, chronic therapy. When treating with an additional therapeutic agent, MRD-containing antibody) can be administered serially, or simultaneously with the additional therapeutic agent.

Therapeutically effective amounts of MRD-containing antibody of the invention vary according to, for example, the targets of the MRD-containing antibody and the potency of conjugated cytotoxic agents encompassed by various embodiments of the invention Thus, for example therapeutically effective dose of an a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) that “mops up” a soluble ligand, such as, TNF alpha, is expected to be higher than that for an a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) that redirects T cell effector function to a target on a hematological malignancy. Likewise, therapeutically effective amounts of a multivalent and multispecific compositions (e.g., MRD-containing antibodies) comprising a maytansinoid cytotoxic agent are likely to be lower than the dosage of an a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) comprising a less potent chemotherapeutic, such as, taxol, or the counterpart a multivalent and monovalent multispecific composition does not contain a cytotoxic agent.

According to one embodiment, a therapeutically effective dose of an a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is an amount selected from about 0.00001 mg/kg to about 20 mg/kg, from about 0.00001 mg/kg to about 10 mg/kg, from about 0.00001 mg/kg to about 5 mg/kg, from about 0.0001 mg/kg to about 20 mg/kg, from about 0.0001 mg/kg to about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 20 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, and from about 0.001 mg/kg to about 5 mg/kg of the patient's body weight, in one or more dose administrations daily, for one or several days.

According to another embodiment, a therapeutically effective amount of an a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is an amount such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (μg) per milliliter (ml) to about 100 μg/ml, from about 1 μg/ml to about 5 μg/ml, and usually about 5 μg/ml. Stated differently, in another embodiment, the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, from about 0.2 mg/kg to about 200 mg/kg, from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

In some embodiments, the a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is administered at about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg to about 5 mg/kg.

In additional embodiments, the interval between dose administration of the multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) is about daily, about twice a week, about every week, about every other week, or about every three weeks. In some embodiments, the multivalent and monovalent multispecific composition is administered first at a higher loading dose and subsequently at a lower maintenance dose.

In further embodiments, therapeutic compositions of the invention comprise multivalent and multispecific compositions (e.g., MRD-containing antibodies) in an amount of at least 0.1 weight percent of antibody per weight of total therapeutic composition. A weight percent is a ratio by weight of antibody/total composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody-MRD per 100 grams of total composition. According to some embodiments, a therapeutic composition comprising a multivalent and monovalent multispecific composition contains about 10 micrograms (μg) per milliliter (ml) to about 100 milligrams (mg) per ml of antibody as active ingredient per volume of composition. In additional embodiments, a therapeutic composition comprising a multivalent and monovalent multispecific composition contains about 1 mg/ml to about 10 mg/ml (i.e., about 0.1 to 1 weight percent) of antibody as active ingredient per volume of composition.

As shown in the examples herein, a multivalent and multispecific composition (e.g., an MRD containing antibody) can have a similar PK profile to a corresponding antibody. Thus, in some embodiments, an antibody-MRD is administered in a dosing concentration and regimen that is the same as the antibody component of the antibody-MRD molecule alone (e.g., a commercial antibody, or a so-called “biosimilar” or a “biobetter” thereof). Likewise, the multivalent and multispecific composition can have a different PK profile from a corresponding antibody. For example, in in embodiments where the multivalent and multispecific compositions redirect a T cell response and/or include a cytotoxic agent, the dosing concentration is expected to be less than that of the corresponding antibody. In these instances, therapeutically effective dosing concentrations and regimens for these compositions can routinely be determined using factors and criteria known in the art.

The multivalent and multispecific compositions (e.g., MRD-containing antibodies) need not be, but optionally are, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of multivalent and monovalent multispecific composition present in the formulation, the type of disorder or treatment, and other factors discussed above.

As discussed above, the appropriate dosage of the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) will depend on the type of disease to be treated, as defined above, the severity and course of the disease, previous therapy, the patient's clinical history, and the discretion of the attending physician. The multivalent and monovalent multispecific composition is suitably administered to the patient at one time or over a series of treatments. Preferably, the multivalent and monovalent multispecific composition is administered by intravenous infusion or by subcutaneous injections. According to some embodiments, the multivalent and monovalent multispecific composition is administered parenterally by injection or by gradual infusion over time. Although the target molecule can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is likelihood that the tissue targeted contains the target molecule. Thus, the multivalent and monovalent multispecific composition can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means. Multivalent and multispecific compositions can also be delivered by aerosol to airways and lungs. In some embodiments, the antibody-MRD molecule is administered by intravenous infusion. In some embodiments, the antibody-MRD molecule is administered by subcutaneous injection.

The therapeutic compositions containing a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) can conventionally be administered intravenously, as by injection of a unit dose, for example. The term “unit dose” when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. In a specific embodiment, the therapeutic compositions containing a human monoclonal antibody or a polypeptide are administered subcutaneously.

The compositions of the invention are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the patient to be treated, capacity of the patient's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

In other embodiments, the invention provides a method for treating or preventing a disease, disorder, or injury comprising administering a therapeutically effective amount or prophylactically effective amount of antibody-MRD molecule to a patient in need thereof. In some embodiments, the disease, disorder or injury is cancer. In other embodiments, the disease, disorder or injury is a disease or disorder of the immune system, such as, inflammation or an autoimmune disease.

Multivalent and multispecific compositions (e.g., MRD-containing antibodies) are expected to have at least the same therapeutic efficacy as the antibody contained in the MRD antibody containing antibody when administered alone. Accordingly, it is envisioned that the multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be administered to a patient to treat or prevent a disease, disorder, or injury for which the antibody contained in the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD-containing antibody, demonstrates a reasonably correlated beneficial activity in treating or preventing such disease, disorder or injury. This beneficial activity can be demonstrated in vitro, in an in vivo animal model, or in human clinical trials. In one embodiment, an MRD-containing antibody is administered to a patient to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD-containing antibody, demonstrates therapeutic or prophylactic efficacy in vitro or in an animal model. In another embodiment, an MRD-containing antibody is administered to a patient to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD-containing antibody, demonstrates therapeutic or prophylactic efficacy in humans. In another embodiment, an MRD-containing antibody is administered to a patient to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD-containing antibody, has been approved by a regulatory authority for use in such treatment or prevention.

In another embodiment, an MRD-containing antibody is administered in combination with another therapeutic to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD antibody, in combination with the therapeutic, or a different therapeutic that functions in the same way as the therapeutic in the combination, demonstrates therapeutic or prophylactic efficacy in vitro or in an animal model. In another embodiment, an MRD-containing antibody is administered in combination with another therapeutic to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD antibody, in combination with the therapeutic, or a different therapeutic that functions in the same way as the therapeutic in the combination, demonstrates therapeutic or prophylactic efficacy in humans. In another embodiment, an MRD-containing antibody, is administered in combination with another therapeutic to treat or prevent a disease, disorder or injury for which the antibody component of the MRD-containing antibody, or an antibody that functions in the same way as the antibody contained in the MRD antibody, in combination with the therapeutic, or a different therapeutic that functions in the same way as the therapeutic in the combination, has been approved by a regulatory authority for use in such treatment or prevention. The administration of an MRD-containing antibody in combination with more than one therapeutic as described above is also encompassed by the invention.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that promotes apoptosis, inhibits apoptosis, promotes cell survival, inhibits cell survival, promotes senescence of diseased or aberrant cells, inhibits cell senescence, promotes cell proliferation, inhibits cell proliferation, promotes cell differentiation, inhibits cell differentiation, promotes cell activation, inhibits cell activation, promotes cell metabolism, inhibits cell metabolism, promotes cell adhesion, inhibits cell adhesion, promotes cell cycling or cell division, inhibits cell cycling or cell division, promotes DNA replication or repair, inhibits DNA replication or repair, promotes transcription or translation, or inhibits transcription or translation.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that promotes apoptosis or senescence of diseased or aberrant cells. In some embodiments, the MRD-containing antibody is administered in combination with a compound that agonizes, antagonizes or reduces the activity of: EGFR, ErbB2, cMET, TNFa, TGFb, integrin αvβ3, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TNFR1, TNFRSF10A (TRAIL R1 DR4), TNFRSF10B (TRAIL R2 DR5), TNF, TRAIL, IFN beta, MYC, Ras, BCR, ABL, JNK, CKH2, CHK1, CDK1, RAC1, MEK, MOS, mTOR, AKT, NFkB, Ikk, IAP1, IAP2, XIAP, b-catenin, survivin, HDAC, HSP70, HSP90, proteasome 20S, topoisomerase 1, MDM2, E2F, or E2F1.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that inhibits cell survival. In some embodiments, the MRD-containing antibody is administered in combination with a compound that antagonizes or reduces the activity of: VEGF, VEGFR1, VEGFR2, IGF1R, IGF1, IGF2, PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D, PDGFRA, PDGFRB, TFGa, TGFB3, PI3K, TNFSF13B (BLYS), TNFRSF13C (BAFFR), JNK, NFKB, SIP, integrin αvβ3, or survivin.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates cell proliferation. In some embodiments, the MRD-containing antibody is administered in combination with a compound that antagonizes or reduces the activity of: VEGF, VEGFR, EGFR, ErbB2, NFKB, HIF, MUC1, MUC2, or HDAC.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates cell adhesion. In some embodiments, the MRD-containing antibody is administered in combination with a compound that inhibits or reduces the activity of: MMP1, MMP2, MMP7, MMP9, MMP12, PLAU, αvβ1 integrin, αvβ3 integrin, αvβ5 integrin, TGFb, EPCAM, α1β1 integrin, α2β1 integrin, α4β1 integrin, α2β1 integrin, α5β1 integrin, α9β1 integrin, α6β4 integrin, αMβ2 integrin, CEA, L1, Me1-CAM, or HIF1. In one embodiment the MRD-containing antibody is administered in combination with a compound that inhibits or reduces the activity of αvβ3 integrin, αvβ5 integrin, or α5β1 integrin. In specific embodiments the MRD-containing antibody is administered in combination with: MEDI-522 (VITAXIN, Abegrin; MedImmune), ATN-161 (Attenuon), EMD 121974 (Merck KGaA), CNTO 95 (Cenotocor), or velociximab (M200, Protein Design Labs).

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates cell activation. In some embodiments, the MRD-containing antibody is administered in combination with a compound that promotes, inhibits or reduces the activity of: CD80, CD86, MHC, PDL2 (B7-DC), B7-H1, B7-H2 (ICOSL), B7-H3, B7-H4, CD28, CTLA4, TCR, PD1, CD80, or ICOS.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates cell cycling, cell division or mitosis. In some embodiments, the MRD-containing antibody is administered in combination with a compound that antagonizes or reduces the activity of: PI3K, SMO, Ptch, HH, SHH, plk1, plk2, plk3, plk4, aurora A, aurora B, aurora C, CDK1, CDK2, CDK4, CHK1, CHK2, GSK3B, PAK, NEK2A, ROCK 2, MDM2, EGF (KSP), proteasome 20S, HDAC, or survivin.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates DNA replication or repair. In some embodiments, the MRD-containing antibody is administered in combination with a compound that antagonizes or reduces the activity of: BRCA1, CHK1, CHK2, E2F, E2FL, MDM2, MDM4, or PARP1.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates transcription or translation. In some embodiments, the MRD-containing antibody is administered in combination with a compound that antagonizes or reduces the activity of: IGF1R, IGF1, IGF2, PDGFRA, PDGFRB, PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D, KIT, MYC, CD28, CDK4, CDK6, mTOR, MDM2, HDAC, E2F, E2F1, or HIF1.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates migration, invasion or metastasis. In some embodiments, the MRD-containing antibody is administered in combination with a compound that inhibits or reduces the activity of: c-MET, RON, CXCR4, PI3K, AKT, MMP2, FN1, CATHD, AMF, αvβ1 integrin, αvβ3 integrin, αvβ5 integrin, TGFb, α1β1 integrin, α2β1 integrin, α4β1 integrin, α2β1 integrin, α5β1 integrin, α9β1 integrin, α6β4 integrin, αMβ2 integrin, or HIF1.

According to one embodiment, an MRD-containing antibody is administered in combination with a compound that regulates cell metabolism. In some embodiments, the MRD-containing antibody is administered in combination with a compound that inhibits or reduces the activity of: ErbB2, EGFR, IGF1R, IGF1, IGF2, TGFa, ICOS, PI3K, VEGFR1, VEGFR2, mTOR, HIF1, or HDAC.

According to one embodiment, an MRD-containing antibody is administered in combination with an inhibitor of one or more protein kinases. In one embodiment, the protein kinase inhibitor inhibits a target of the MRD containing antibody (e.g., by either one or more MRDs or the antibody of the MRD containing antibody). In an alternative embodiment, the protein kinase inhibitor inhibits a protein kinase that is not a target of the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits one protein kinase. In other embodiments, the protein kinase inhibitor inhibits more than one protein kinase.

In some embodiments, an MRD containing antibody is administered in combination with an inhibitor (e.g., small molecule, antibody, etc.,) of a protein kinase selected from: EGFR, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, ErbB2, VEGFR1, VEGFR2, VEGFR3, Tie-2, PDGFR, PDGFRB, RON, and c-Met. In other embodiments, the inhibitor inhibits a protein kinase that is not targeted by the MRD containing antibody. In an additional embodiment, an MRD-containing antibody is administered in combination with an inhibitor of one or more protein kinases selected from: EGFR, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, ErbB2, VEGFR1, VEGFR2, VEGFR3, Tie-2, PDGFRA, PDGFRB, FIT3, ALK, RET, Kit, raf, p38, RON, c-Met, PI3K, ERK, FAK, AKT, SYK, JAK1, JAK2, JAK3, TYK2, SIP, FAK, PTK7, PKD1, PKA, PKC, PKG, PRKDC, Pim, CDK, plk, p38MAPK, SRC, ABL, FGR, FYN, HCK, LCK, LYN, YES, EPH4, BMK1, ERK5, mTOR, CHK1, CHK2, CSNK1G1, CSNK1G2, CSNK1G3, GSK3, BTK, JNK, Aurora Kinase, Aurora Kinase A, Aurora Kinase B, and Aurora Kinase C.

In an additional embodiment, an MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: imatinib mesylate (e.g., GLEEVEC™), gefitinib (e.g., IRESSA™, Astra Zeneca), vandetanib (e.g., ZACTIMA™, Astra Zeneca), erlotinib (e.g., TARCEVA™, Genentech/OSI), sunitinib (e.g., SUTENT™, Pfizer), lapatanib (GSK), and sorafenib (e.g., NEXAVAR™, Bayer).

In a further embodiment, an MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: nilotinib (e.g., AMN107, Novartis), dasatinib (e.g., BMS 354825, BMS), ABT-869, botsutinib (e.g., SKI-606, Wyeth), cediranib, recentib, captastatin, AEE788 (Novartis), AZD0530 (AstraZeneca) Exel 7646/Exel 0999 Exelixis), cabozantinib (e.g., XL184; Exelixis), XL880/GSK1363089 (Exelixis/GSK), ARQ-197 (Arqule and Daiichi Sankyo), Inno-406 (Innovive), SGS523 (SGX), PF-2341066 (Pfizer), CI-1033 (Pfizer), motesanib (e.g., AMG-706, Amgen), AG-013736 (Axitinib), AMG-705 (Amgen), pegaptanib (OSI/Pfizer), lestaurtinib, ruxolitinib, SB1518, CYT387, LY3009104, TG101348 JANEX-1, tofacitinib (Pfizer), INCB18424, LFM-A13, pazopanib (e.g., GW786034B, GlaxoSmithKline), GW-572016, EKB-569 (Wyeth-Ayerst), vatalanib (e.g., PTK787/ZK), AZD2171, MK-0457 (VX-680, Merck), PHA 739358 (Nerviano), mubritinib (Takeda), E7080 (Eisai), fostamatinib (Rigel/AstraZeneca), SGX523, SNS-032 (Sunesis), XL143, SNS-314 (Sunesis), SU6668 (Pfizer), AV-951 (AVEO), AV-412 (AVEO), tivizanib (AVEO), PX-866 (Oncothyreon), canertinib (CI-1033), NSC 109555, VRX0466617, UCN-01, CHK2 inhibitor II, EXEL-9844, XL844, CBP501, PF-004777736, debromohymerialdisine, Go6976, AEG3482, cediranib (e.g., RECENTIN™, AstraZeneca), semaxanib (SU5416), SU5616, CGP, 53716, mastinib, and ZD6474 (AstraZeneca).

In a further embodiment, an MRD-containing antibody is administered in combination with a FGFR protein kinase inhibitor selected from: sunitinib, SU5402, PD173074, TKI258 (Novartis), BIBF 1120 (Boehringer Ingelheim), brivanib (BMS-582,664), E7080 (Eisai), and TSU-68 (Taiho).

In an additional embodiment, an MRD-containing antibody is administered in combination with a protein kinase inhibitor of JAK1, JAK2, JAK3, or SYK. In a further embodiment the protein kinase inhibitor is selected from: lestaurtinib, tofacitinib, ruxolitinib, SB1518, CYT387, LY3009104, TG101348, fostamatinib, BAY 61-3606, and sunitinib.

In one embodiment, an ErbB2 (HER2) binding MRD-containing antibody (e.g., an MRD-binding antibody that binds ErbB2 by either one or more MRDs or the antibody of the MRD containing antibody) is administered in combination with a protein kinase inhibitor of ErbB2. In another specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with a protein kinase inhibitor of ErbB2. In one embodiment, an ErbB2-binding MRD-containing antibody is administered in combination with lapatinib. In a specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with lapatinib. In one embodiment, an ErbB2-binding MRD-containing antibody is administered in combination with sunitinib. In a specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with sunitinib. In one embodiment, an ErbB2-binding MRD-containing antibody is administered in combination with neratinib. In a specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with neratanib. In one embodiment, an ErbB2-binding MRD-containing antibody is administered in combination with iapatinib. In a specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with iapatinib. In an additional embodiment, an ErbB2 (HER2) binding MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: canertinib (GW-572016), AV-412 (AVEO), tivozanib (AVEO), vandetanib (e.g., ZACTIMA™, AstraZeneca), AEE788 (Novartis), Exel 7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), and EKB-569 (Wyeth-Ayerst). In a specific embodiment a trastuzumab antibody-based MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: canertinib (GW-572016), AV-412 (AVEO), tivozanib (AVEO), vandetanib (e.g., ZACTIMA™, AstraZeneca), AEE788 (Novartis), Exel 7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), PX-866 (Oncothyreon), and EKB-569 (Wyeth-Ayerst).

In another embodiment, an EGFR binding MRD-containing antibody (e.g., an MRD-binding antibody that binds EGFR by either one or more MRDs or the antibody of the MRD containing antibody) is administered in combination with a protein kinase inhibitor of EGFR. In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with a protein kinase inhibitor of EGFR. In one embodiment, an EGFR binding MRD-containing antibody is administered in combination with gefitinib (e.g., IRESSA™, AstraZeneca). In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with gefitinib (e.g., IRESSA™, AstraZeneca). In one embodiment, an EGFR binding MRD-containing antibody is administered in combination with erlotinib (e.g., TARCEVA™, Genentech/OSI). In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with erlotinib (e.g., TARCEVA™, Genentech/OSI). In one embodiment, an EGFR binding MRD-containing antibody is administered in combination with lapatinib. In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with lapatinib. In one embodiment, an EGFR binding MRD-containing antibody is administered in combination with sorafenib (e.g., NEXAVAR™, Bayer). In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with sorafenib (e.g., NEXAVAR™, Bayer). In another embodiment, an EGFR binding MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: canertinib (GW-572016), ZD6474, AV-412 (AVEO), tivozanib (AVEO), vandetanib (ZACTIMA, AstraZeneca), AEE788 (Novartis), Exel 7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), and EKB-569 (Wyeth-Ayerst). In a specific embodiment a cetuximab antibody-based MRD-containing antibody is administered in combination with a protein kinase inhibitor selected from: canertinib (GW-572016), ZD6474, AV-412 (AVEO), tivozanib (AVEO), vandetanib (ZACTIMA, AstraZeneca), AEE788 (Novartis), Exel 7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), PX-866 (Oncothyreon), and EKB-569 (Wyeth-Ayerst).

In one embodiment, a VEGFA, VEGFR1, or VEGFR2 binding MRD-containing antibody (e.g., an MRD-binding antibody that binds VEGFR1 by either one or more MRDs or the antibody of the MRD containing antibody) is administered in combination with a protein kinase inhibitor of VEGR1, VEGFR2, or VEGFR3. In one embodiment, the VEGFA, VEGFR1 or VEGFRr2 binding MRD-containing antibody is administered in combination with: sunitinib, sorafenib, pazopanib (e.g., GW786034B), AZD2171, vatalanib, ZD6474, AMG-706, or AC013736.

In a further embodiment, an MRD-containing antibody is administered in combination with a proteasome inhibitor. In a specific embodiment, the inhibitor is bortezomib (e.g., VELCADE™). In another specific embodiment, the inhibitor is PR-171 (Proteolix).

In a further embodiment, an MRD-containing antibody is administered in combination with a HDAC inhibitor.

In a further embodiment, an MRD-containing antibody is administered in combination with a mTOR inhibitor.

In a further embodiment, an MRD-containing antibody is administered in combination with a NFKB inhibitor.

In one embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of a VEGFA or VEGFR binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating colorectal cancer by administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having colorectal cancer. In another embodiment, the invention provides a method of treating breast cancer by administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having breast cancer. In another embodiment, the invention provides a method of treating non-small cell lung carcinoma by administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having non-small cell lung carcinoma. In other embodiments, therapeutic effective amounts of bevacizumab comprising at least one MRD are administered to a patient to treat metastatic colorectal cancer, metastatic breast cancer, metastatic pancreatic cancer, or metastatic non-small cell lung carcinoma. In another embodiment, the invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having renal cell carcinoma, glioblastoma multiforme, ovarian cancer, prostate cancer, liver cancer or pancreatic cancer.

Combination therapy and compositions including multivalent and multispecific compositions (e.g., MRD-containing antibodies) of the invention and another therapeutic are also encompassed by the invention, as are methods of treatment using these compositions. In other embodiments, compositions of the invention are administered alone or in combination with one or more additional therapeutic agents. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the therapeutic compounds or agents given first, followed by the second. Accordingly, in one embodiment, a VEGFA or VEGFR binding MRD-containing antibody is administered in combination with 5-fluorouracil, carboplatin, paclitaxel, or interferon alpha. In another embodiment, bevacizumab comprising at least one MRD is administered in combination with 5-fluorouracil, carboplatin, paclitaxel, or interferon alpha.

In another embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of a VEGFA or VEGFR binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of ranibizumab comprising at least one MRD to a patient in need thereof.

In some embodiments, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of an ErbB2 (HER2) binding MRD-containing antibody to a patient in need thereof. In various embodiments, the ErbB2-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have been previously shown to respond to another ErbB2-based therapy (e.g., HERCEPTIN, chemotherapy and/or radiation) or are predicted to respond to another ErbB2-based therapy. In other embodiments, the ErbB2-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have previously failed to respond to another ErbB2-based therapy or are predicted to fail to respond to another ErbB2-based therapy.

In a specific embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of trastuzumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating breast cancer by administering a therapeutically effective amount of trastuzumab comprising at least one MRD to a patient having breast cancer. In other embodiments, therapeutic effective amounts of trastuzumab comprising at least one MRD are administered to a patient to treat metastatic breast cancer.

In another embodiment, an ErbB2 (HER2) binding MRD-containing antibody is administered in combination with cyclophosphamide, paclitaxel, docetaxel, carboplatin, anthracycline, or a maytansinoid. In a specific embodiment, trastuzumab comprising at least one MRD is administered in combination with cyclophosphamide, paclitaxel, docetaxel, carboplatin, anthracycline, or a maytansinoid.

In another embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of a CD20-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a hematologic cancer comprising administering a therapeutically effective amount of rituximab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating CD20 positive NHL by administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having CD20 positive NHL. In one embodiment, the invention provides a method of treating CD20 positive CLL by administering a therapeutically effective amount of bevacizumab comprising at least one MRD to a patient having CD20 positive CLL.

In another embodiments, a therapeutically effective amount of a CD20-binding MRD-containing antibody is administered in combination with: ludarabine, cyclophosphamide, FC (fludarabine and cyclophosphamide), anthracycline based chemotherapy regimen (e.g., CHOP (cyclophosphamide, adriamycin, vincristine and prednisone)), or CVP (cyclophosphamide, prednisone, and vincristine) chemotherapy. In a specific embodiment, a therapeutically effective amount of bevacizumab comprising at least one MRD is administered in combination with: ludarabine, cyclophosphamide, FC (fludarabine and cyclophosphamide), anthracycline based chemotherapy regimen (e.g., CHOP (cyclophosphamide, adriamycin, vincristine and prednisone)), or CVP (cyclophosphamide, prednisone, and vincristine) chemotherapy.

Any of the antibody-MRD fusions containing antibodies and/or MRDs that bind CD20 can be used according to the methods of treating a disorder associated with CD20, or that can be treated by targeting cells that express CD20 (e.g., hematological cancers and autoimmune disease). In some embodiments, the antibody component of the antibody-MRD-fusion is selected from rituximab, ocrelizumab, GA101, and PF-5,230,895.

The invention also provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody. In some embodiments, the administered MRD-containing antibody binds a target selected from: CD20, TNFRSF5 (CD40), CD45RB, CD52, CD200, CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5 (CD40 LIGAND), TLR2, TLR4, GPR44, FASL, TREM1, IL1, IL1 beta, IL1RN, tissue factor, MIF, M1P2, 1L6, 1L8, IL10, 1L12, IL13, IL15, IL17, IL18, IL23, TNF, TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN alpha, IFN gamma, GMCSF, FGF, TGFb, C5, and CCR3. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention.

In particular embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody that binds TNF and ANG2.

In additional embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody that binds IL1, IL12, and TNF. In further embodiments, the MRD-containing antibody binds IL1, IL12, TNF and ANG2.

In additional embodiments, the administered MRD-containing antibody binds IL1, IL6 and TNF. In further embodiments, the MRD-containing antibody binds IL1, IL6, TNF and ANG2.

target selected from: CD20, TNFRSF5 (CD40), CD45RB, CD52, CD200, CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5 (CD40 LIGAND), TLR2, TLR4, GPR44, FASL, TREM1, IL1, IL1 beta, IL1RN, tissue factor, MIF, MIP2, IL6, IL8, IL10, IL12, IL13, IL15, IL17, IL18, IL23, TNF, TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN alpha, IFN gamma, GMCSF, FGF, TGFb, C5, and CCR3. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind 2, 3, 4, 5 or more of these targets are also encompassed by the invention.

In additional embodiments, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of an MRD-containing antibody. In a specific embodiment, the administered MRD-containing antibody binds a target selected from: CD1C, CD3, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD28, CD37, CD38, CD45RB, CD52, CD69, CD72, CD74, CD75, CD79A, CD79B, CD80, CD81, CD83, CD86, CD200, IL2RA, IL1R2, IL6R, VLA4, HLA-DRA, HLA-A, ITGA2, ITGA3, CSF2, TLR2, TLR4, GPR44, TREM1, TIE2, TNF, FASL, tissue factor, MIF, MIP2, IL1, IL1 beta, IL1RN, IL2, IL4, IL6, IL8, IL10, IL11, IL12, IL13, IL15, IL17, IL18, IL23, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, ICOSL (B7-H2), MS4A1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70), PAFR, INHA, INHBA, DPP4, NT5E, CTLA4, B7.1/B7.2, LPS, VEGF, GMCSF, FGF, C5, CXCL13, CXCR4, CCR2 and CCR3. In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat rheumatoid arthritis and the multivalent and multispecific compositions bind a target selected from: CD19, CD20, CD45RB, CD52CD200, IL1, IL6, IL12, IL15, IL17, IL18, IL23, TNF, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), TNFSF13B (BLyS), VEGF, VLA4, IFN gamma, IFN alpha, GMCSF, FGF, C5, CXCL13 and CCR2. In additional embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat systemic lupus erythematous and the multivalent and multispecific compositions bind IFN alpha and TNFSF13B (BLyS). In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat multiple sclerosis and the multivalent and multispecific compositions bind a target selected from: ANG2, IL1, IL12, IL18, IL23, CXCL13, TNF, TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma, GM-CSF, FGF, C5, CD52, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFRSF21 (DR6), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, AGE (S100 A, amphoterin), ICOSL (B7-H2), MS4A 1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70), MCP1, CCR2 and CXCL13. Multivalent and multispecific compositions that bind at least 2, 3, 4, 5 or more of these targets are also encompassed by the invention.

In a further embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a CD20-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of a CD20-binding MRD-containing antibody to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of a rituximab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of a rituximab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a rituximab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of a rituximab-MRD-containing antibody to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of an ocrelizumab-MRD-containing antibody to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of an ocrelizumab-MRD-containing antibody to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a ocrelizumab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of an ocrelizumab-MRD-containing antibody to a patient in need thereof.

In another embodiment, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of a PF5,230,895-MRD-containing antibody to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of a PF5,230,895-MRD-containing antibody to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a PF5,230,895-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of an PF5,230,895-MRD-containing antibody to a patient in need thereof.

In some embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody that binds CD20. In further embodiments, the administered MRD-containing antibody binds CD20 and a target selected from: TNF, TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), TNFSF12 (TWEAK), TNFRSF1A, CD45, RB, CD52, CD200, CCR2, PAFR, IL6R, VLA4, CSF2, RAGE, TLR2, TLR4, GPR44, FASL, TREM1, TIE2, tissue factor, MIF, MIP2, LPS, IL1, IL1 beta, IL1RN, IL6, IL6R, IL8, IL10, IL12, IL13, IL15, IL17, IL18, IL23, CXCL13, VEGF, IFN alpha, IFN gamma, GMCSF, FGF, C5, and CCR3. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind CD20 and also bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD20. In further embodiments, the antibody component of the MRD-containing antibody is a rituximab, ocrelizumab, GA101 or PF-5,230,895.

In some embodiments, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of an MRD-containing antibody that binds CD20. In a specific embodiment, the administered MRD-containing antibody binds CD20 and a target selected from: CD1C, CD3, CD4, CD19, CD21, CD22, CD23, CD24, CD28, CD37, CD38, CD45RB, CD52, CD69, CD72, CD74, CD75, CD79A, CD79B, CD80, CD81, CD83, CD86, CD200, IL2RA, IL1R2, IL6R, VLA4, HLA-DRA, HLA-A, ITGA2, ITGA3, CSF2, TLR2, TLR4, GPR44, TREM1, TIE2, TNF, FASL, tissue factor, MIF, MIP2, IL1, IL1 beta, IL1RN, IL2, IL4, IL6, IL8, IL10, IL11, IL12, IL13, IL15, IL17, IL18, IL23, TIE2, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, ICOSL (B7-H2), MS4A1, IFN alpha, IFN IFN gamma, TNFSF7 (CD27 Ligand, CD70), PAFR, INHA, INHBA, DPP4, NT5E, CTLA4, B7.1/B7.2, LPS, VEGF, GMCSF, FGF, C5, CXCL13, CXCR4, CCR2 and CCR3. In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat rheumatoid arthritis and the multivalent and multispecific compositions bind CD20 and a target selected from: CD19, CD45RB, CD52, CD200, IL12, IL15, IL17, IL18, IL23, TNF, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), VEGF, VLA4, IFN gamma, interferon alpha, GMCSF, FGF, C5, CXCL13 and CCR2. In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat multiple sclerosis and the multivalent and multispecific compositions bind CD20 and a target selected from: ANG2, 1L12, 1L18, 1L23, CXCL13, TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma, GM-CSF, FGF, C5, CD52, TIE2, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFRSF21 (DR6), TNFSF12 (TWEAK), TNFSF13B (BLyS), ICOSL (B7-H2), MS4A 1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70), CCR2 and CXCL13. Multivalent and multispecific compositions that bind a least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNF. In further embodiments, the antibody component of the MRD-containing antibody is selected from rituximab, ocrelizumab, GA101 and PF-5,230,895.

In some embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a TNF-binding MRD-containing antibody to a patient in need thereof. In various embodiments, the TNF-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have been previously shown to respond to another TNF-based therapy or are predicted to respond to another TNF-based therapy (e.g., TNF antagonists such as, anti-TNFs (e.g., HUMIRA), EMBREL, CD28 antagonists, CD20 antagonists, and IL6/1L6R antagonists). In other embodiments, the TNF-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have previously failed to respond to another TNF-based therapy or are predicted to fail to respond to another TNF-based therapy.

In some embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody that binds TNF.

In further embodiments, the administered MRD-containing antibody binds TNF and a target selected from: CD20, TNFRSF5 (CD40), CD45RB, CD52, CD200, CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5 (CD40 LIGAND), TLR2, TLR4, GPR44, FASL, TREM1, IL1, IL1 beta, IL1RN, tissue factor, MIF, MIP2, IL6, IL8, IL10, IL12, IL13, IL15, IL17, IL18, IL23, TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN gamma, GMCSF, FGF, C5, and CCR3. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TNF and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNF. In further embodiments, the antibody component of the MRD-containing antibody is selected from adalimumab, certolizumab, golimumab and AME-527.

In some embodiments, the invention provides a method of treating an autoimmune disease comprising administering a therapeutically effective amount of an MRD-containing antibody that binds TNF. In a specific embodiment, the administered MRD-containing antibody binds TNF and a target selected from: CD1C, CD3, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD28, CD37, CD38, CD45RB, CD52, CD69, CD72, CD74, CD75, CD79A, CD79B, CD80, CD81, CD83, CD86, CD200, IL2RA, IL1R2, IL6R, VLA4, HLA-DRA, HLA-A, ITGA2, ITGA3, CSF2, TLR2, TLR4, GPR44, TREM1, TIE2, FASL, tissue factor, MIF, MIP2, IL1, IL1 beta, IL1RN, IL2, 1L4, IL6, IL8, IL10, IL11, IL12, IL13, IL15, IL17, IL18, IL23, TIE2, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, ICOSL (B7-H2), MS4A1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70), PAFR, INHA, INHBA, DPP4, NT5E, CTLA4, B7.1/B7.2, LPS, VEGF, GMCSF, FGF, C5, CXCL13, CXCR4, CCR2 and CCR3. In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat rheumatoid arthritis and the multivalent and multispecific compositions bind TNF and a target selected from: CD19, CD20, CD45RB, CD52CD200, IL12, IL15, IL17, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), TNFSF13B (BLyS), VEGF, VLA4, IFN gamma, interferon alpha, GMCSF, FGF, C5, CXCL13 and CCR2. In further embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to treat multiple sclerosis and the multivalent and multispecific compositions bind TNF and a target selected from: ANG2, IL12, IL18, IL23, CXCL13, TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma, GM-CSF, FGF, C5, CD52, TNFRSF1A, TNFRSF5 (CD40), TIE2, TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFRSF21 (DR6), TNFSF12 (TWEAK), TNFSF13B (BLyS), ICOSL (B7-H2), MS4A 1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70), CCR2 and CXCL13. Multivalent and multispecific compositions that bind a least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TNF. In further embodiments, the antibody component of the MRD-containing antibody selected from adalimumab, certolizumab, golimumab and AME-527.

In other embodiments, the TNF-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have been previously shown to respond to an autoimmune disease based therapy or are predicted to respond to other autoimmune disease based therapies (e.g., TNF antagonists such as, Anti-TNFs (e.g., HUMIRA®), ENBREL®, CD28 antagonists, CD20 antagonists, BLyS antagonists, and IL6/IL6R antagonists). In other embodiments, the TNF-binding multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to patients who have previously failed to respond to another autoimmune disease based therapy or are predicted to fail to respond to another autoimmune disease based therapy.

In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of adalimumab comprising at least one MRD to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating an autoimmune disease, by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of ATN-103 comprising at least one MRD to a patient in need thereof.

In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease, by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of infliximab comprising at least one MRD to a patient in need thereof.

In some embodiments, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of an MRD-containing antibody that binds TNFSF15 (TL1A).

In further embodiments, the administered MRD-containing antibody binds TL1A and a target selected from: TNF, IFN gamma, IL1, IL1beta, IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32. Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind TL1A and at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds TL1A.

In an additional embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a IL22-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of PF5,212,367 (ILV-094) comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of PF5,212,367 comprising at least one MRD to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a alpha4 integrin-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a natalizumab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of a natalizumab-MRD-containing antibody to a patient in need thereof. In a further embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis, by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of natalizumab comprising at least one MRD to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a TNFSF5 (CD40 LIGAND)-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a CDP7657-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of a CDP7657-MRD-containing antibody to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of CDP7657 comprising at least one MRD to a patient in need thereof.

In another embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a TNFSF12 (TWEAK)-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody (that has entered phase 1 clinical trials) comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of a CD25-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating a disorder of the immune system comprising administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating systemic lupus erythematous comprising administering a therapeutically effective amount of a daclizumab-MRD-containing antibody to a patient in need thereof. In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of a daclizumab-MRD-containing antibody to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In a further embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof. In an additional embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of daclizumab comprising at least one MRD to a patient in need thereof.

Antibody-MRD fusion proteins having antibodies and/or MRDs that bind cancer antigens or other targets associated with cancer establishment, progression, and/or metastasis are described herein or otherwise known in the art and may be used according to the methods of the invention to treat cancer. In specific embodiments the antibody-MRD fusion proteins comprise an antibody and/or MRD that bind to a target identified herein.

In another embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of an EGFR-binding MRD-containing antibody to a patient in need thereof. In a specific embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of cetuximab comprising at least one MRD to a patient in need thereof. In one embodiment, the invention provides a method of treating cancer by administering a therapeutically effective amount of cetuximab comprising at least one MRD to a patient having colorectal cancer. In another embodiment, therapeutic effective amounts of cetuximab comprising at least one MRD are administered to a patient to treat metastatic colorectal cancer, metastatic breast cancer, metastatic pancreatic cancer, or metastatic non-small cell lung carcinoma. In one embodiment, the invention provides a method of treating cancer by administering a therapeutically effective amount of cetuximab comprising at least one MRD to a patient having squamous cell carcinoma of the head and neck.

In another embodiment, a therapeutically effective amount of an EGFR-binding MRD-containing antibody is administered in combination with irinotecan, FOLFIRI, platinum-based chemotherapy, or radiation therapy. In a specific embodiment, a therapeutically effective amount of cetuximab comprising at least one MRD is administered in combination with irinotecan, FOLFIRI, platinum-based chemotherapy, or radiation therapy.

In certain embodiments, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of an MRD-antibody described herein to a patient in need thereof.

In one embodiment, the invention provides a method of treating a solid cancer by administering a therapeutically effective amount of a solid cancer binding MRD-antibody described herein (e.g., an MRD-antibody that binds a validated solid tumor associated target as described herein to a patient in need thereof.

In some embodiments, the invention provides a method of treating a solid cancer by administering a therapeutically effective amount of an MRD-antibody that binds to a member selected from the group consisting of: IGFR1, ALK1, p-cadherin, CRYPTO, and alpha5 b1 integrin. In other embodiments, the antibody component of the administered MRD-antibody is a member selected from: figitumumab, CP-870893, PF-3,732,010, PF-3,446,962, volociximab, BIIB022, and the Biogen CRYPTO antibody.

In some embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) described herein are useful for treating cancer. Thus, in some embodiments, the invention provides methods of treating cancer comprise administering a therapeutically effective amount of a MRD-containing antibody to a patient (e.g., a patient (subject) in need of treatment). In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the patient is a human.

Other examples of cancers or malignancies that may be treated with MRD containing antibodies and MRDs include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extra cranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extra cranial Germ Cell Tumor, Extra gonadal Germ Cell Tumor, Extra hepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, and Wilms' Tumor.

In some embodiments, multivalent and multispecific compositions (e.g., MRD-containing antibodies) are useful for inhibiting tumor growth. In certain embodiments, the method of inhibiting the tumor growth comprises contacting the cell with a MRD-containing antibody in vitro. For example, an immortalized cell line or a cancer cell line that expresses an MRD target and/or an antibody target is cultured in medium to which is added the MRD-containing antibody to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample for example, a tissue biopsy, pleural effusion, or blood sample and cultured in medium to which is added a MRD-containing antibody to inhibit tumor growth.

In some embodiments, the method of inhibiting tumor growth comprises contacting the tumor or tumor cells with a therapeutically effective amount of the MRD-containing antibody in vivo. In certain embodiments, contacting a tumor or tumor cell is undertaken in an animal model. For example, multivalent and multispecific compositions (e.g., MRD-containing antibodies) can be administered to xenografts in immunocompromised mice (e.g., NOD/SCID mice) to inhibit tumor growth. In some embodiments, cancer stem cells are isolated from a patient sample for example, a tissue biopsy, pleural effusion, or blood sample and injected into immunocompromised mice that are then administered a MRD-containing antibody to inhibit tumor cell growth. In some embodiments, the MRD-containing antibody is administered at the same time or shortly after introduction of tumorigenic cells into the animal to prevent tumor growth. In some embodiments, the MRD-containing antibody is administered as a therapeutic after the tumorigenic cells have grown to a specified size.

In certain embodiments, the method of inhibiting tumor growth comprises administering to a patient (subject) a therapeutically effective amount of a MRD-containing antibody. In certain embodiments, the patient is a human. In certain embodiments, the patient has a tumor or has had a tumor removed. In certain embodiments, the tumor expresses an antibody target. In certain embodiments, the tumor overexpresses the MRD target and/or the antibody target.

In certain embodiments, the inhibited tumor growth is selected from the group consisting of brain tumor, colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a breast tumor.

In additional embodiments, multivalent and multispecific compositions (e.g., MRD-containing antibodies) are useful for reducing tumorigenicity. Thus, in some embodiments, the method of reducing the tumorigenicity of a tumor in a patient, comprises administering a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) to the patient. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody).

In other embodiments, multivalent and multispecific compositions (e.g., MRD-containing antibodies) are useful for diagnosing, treating or preventing a disorder of the immune system. In one embodiment, the disorder of the immune system is inflammation or an inflammatory disorder. In a more specific embodiment, the inflammatory disorder is selected from the group consisting of asthma, allergic disorders, and rheumatoid arthritis. In further embodiment, the disorder of the immune system is an autoimmune disease. Autoimmune disorders, diseases, or conditions that may be diagnosed, treated or prevented using multivalent and multispecific compositions (e.g., MRD-containing antibodies) include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmia, polyendocrinopathies, purpura (e.g., Henloch-scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erythematous, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as, primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders.

In another embodiment the disorder of the immune system diagnosed, treated or prevented using multivalent and multispecific compositions (e.g., MRD-containing antibodies) is selected from the group consisting of: Crohn's disease, Systemic lupus erythematous (SLE), inflammatory bowel disease, psoriasis, diabetes, ulcerative colitis, multiple sclerosis, and rheumatoid arthritis. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis.

In other embodiments, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is administered to a patient to treat a metabolic disease or disorder.

In other embodiments, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is administered to a patient to treat a cardiovascular disease or disorder. In one embodiment, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) is administered to a patient to treat thrombosis, atherosclerosis, heart attack, or stroke.

In another embodiments, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibodies) is administered to a patient to treat a musculoskeletal disease or disorder.

In further embodiments, a therapeutically effective amount of a multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is administered to a patient to treat a skeletal disease or disorder. In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is administered to a patient to treat osteoporosis.

In additional embodiments, the multivalent and monovalent multispecific composition binds (1) a target on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell) and (2) a target on a leukocyte, such as, a T-cell receptor molecule. According to one embodiment, the binding of one or more targets by the multivalent and monovalent multispecific composition is used to direct an immune response to an infectious agent, cell, tissue, or other location of interest in a patient. For example, in some embodiments an MRD of the multivalent and monovalent multispecific composition binds a target on the surface of an effector cell. Thus, in some embodiments, an MRD of the multivalent and monovalent multispecific composition binds a target on the surface of a T cell. In specific embodiments an MRD of the multivalent and monovalent multispecific composition binds CD3. In other embodiments, an MRD of the multivalent and monovalent multispecific composition binds CD2. In further embodiments, an MRD of the multivalent and monovalent multispecific composition binds the T-cell receptor (TCR). According to additional embodiments, an MRD of the multivalent and monovalent multispecific composition binds a target on the surface of a Natural Killer Cell. Thus, in some embodiments, an MRD of the multivalent and monovalent multispecific composition binds a NKG2D (Natural Killer Group 2D) receptor. In additional embodiments an MRD of the multivalent and monovalent multispecific composition binds CD16 (i.e., Fc gamma RIII) CD64 (i.e., Fc gamma RI), or CD32 (i.e., Fc gamma RII). In additional embodiments, the multispecific composition contains more than one monospecific binding site for different targets.

Thus, in some embodiments, a multivalent and monovalent multispecific composition (e.g., an MRD-containing antibody) binds a target on a leukocyte and a tumor antigen on a tumor cell. In some embodiments, the MRD-containing antibody binds NKG2D. In further embodiments, an MRD-containing antibody binds NKG2D and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and EPCAM. In some embodiments, the MRD-containing antibody binds CD3. In particular embodiments, the MRD-containing antibody binds CD3 epsilon. In further embodiments, an MRD-containing antibody binds CD3 and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and EPCAM. In some embodiments, the MRD-containing antibody binds CD4. In further embodiments, an MRD-containing antibody binds CD4 and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and EPCAM.

In further embodiments, the multivalent and monovalent multispecific composition has a single binding site (i.e., is monospecific) for a target. In some embodiments, the multivalent and monovalent multispecific composition has a single binding site (i.e., is monospecific) for a target on a leukocyte, such as, a T-cell (e.g., CD3) and binds a target on a cell or tissue of interest (e.g., a tumor antigen on a tumor cell, such as, a target disclosed herein).

In further embodiments, the invention is directed to treating a disease or disorder by administering a therapeutically effective amount of a multivalent and monovalent multispecific composition that has a single binding site (i.e., is monospecific) for a target. In some embodiments, the administered multivalent and monovalent multispecific composition has a single binding site (i.e., is monospecific) for a target on a leukocyte, such as, a T-cell (e.g., CD3) and binds a target on a cell or tissue of interest (e.g., a tumor associated antigen on a tumor cell). In some embodiments, the tumor cell is from a cancer selected from breast cancer, colorectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer, pancreatic cancer, and thyroid cancer.

Additional embodiments are directed to administering a therapeutically effective amount of a multivalent and monovalent multispecific composition to treat a neurological disease or disorder selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g., HIV-associated dementia). In another embodiment, the multivalent and monovalent multispecific composition is administered to a patient to treat a brain cancer, metastatic cancer of the brain, or primary cancer of the brain. In a further embodiment, the multivalent and monovalent multispecific composition is administered to a patient to treat brain injury, stroke, spinal cord injury, or pain management. In further embodiments, the multivalent and monovalent multispecific composition is administered to a patient to treat brain injury, stroke, or spinal cord injury, or for pain management.

In one embodiment, a therapeutically effect amount of the multivalent and monovalent multispecific composition is administered to a patient to treat an infection or a symptom associated with an infection caused by an infectious agent. In some embodiments, the infection is caused by a member selected from apovavirus (e.g., JC polyomavirus), trypanosomes, West Nile virus, HIV, Streptococcus pneumoniae and Haemophilus influenzae, bovine spongiform encephalopathy, meningitis, Progressive multifocal leukoencephalopathy (PML), Late-stage neurological trypanosomiasis, Encephalitis, and rabies.

According to some embodiments, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) is able to cross the blood brain barrier (BBB) and bind a target located on the brain side of the BBB. In additional embodiments, the multivalent and monovalent multispecific composition has a single binding site that binds a target (e.g., ligand, receptor, or accessory protein) associated with an endogenous BBB receptor mediated transport system. In some embodiments, a single binding site of the composition is an MRD. In other embodiments, a single binding site of the composition is an antibody antigen binding domain. In some embodiments, the multivalent and monovalent multispecific composition contains 1, 2, 3, 4, 5, or more single binding sites (i.e., monovalently binds) for a target associated with an endogenous BBB receptor mediated transport system and the composition is able to cross to the cerebrospinal fluid side of the BBB. In additional embodiments, the multivalent and monovalent multispecific composition contains 1, 2, 3, 4, 5, or more multiple binding sites (i.e., multivalently binds) for a target associated with an endogenous BBB receptor mediated transport system and the composition is able to cross to the cerebrospinal fluid side of the BBB. In additional embodiments, a therapeutically effective amount of an MRD-containing antibody is administered to a patient to treat a neurological disease or disorder selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, neuromyelitis optica and beuro-AIDS (e.g., HIV-associated dementia). In some embodiments, the multivalent and monovalent multispecific composition has a single binding site (i.e., is monovalent for binding a particular target (antigen)) or two or more binding sites (i.e., is monovalent for binding a particular target) for a target selected from alpha-synuclein, RGM A, NOGO A, NgR, OMGp MAG, CSPG, neurite inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4) an ephrin, A-beta, AGE (S100 A, amphoterin), NGF, soluble A-B, aggrecan, midkine, neurocan, versican, phosphacan, Te38 and PGE2. In some embodiments, the multivalent and monovalent multispecific composition additionally has a single binding site or multiple binding sites for a target selected from IL1, IL1R, IL6, IL6R, IL12, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), CD45RB, CD52, CD200, VEGF, VLA4, TNF alpha, Interferon gamma, GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a, and MCP-1.

In additional embodiments, the multivalent and monovalent multispecific composition is capable of transferring to the cerebrospinal fluid side of the BBB and is administered to a patient to treat a neurological disease or disorder selected from: brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, neuromyelitis optica and neuro-AIDS (e.g., HIV-associated dementia). In further embodiments, the invention is directed to treating a disease or disorder by administering an MRD-containing antibody that has a single binding site (i.e., is monospecific) for a target to a patient in need thereof. In some embodiments, the administered MRD-containing antibody has a single binding site (i.e., is monospecific) for a target on a leukocyte, such as, a T-cell (e.g., CD3) and binds a target on a cell or tissue of interest (e.g., a tumor associated antigen on a tumor cell).

In some embodiments, the multivalent and monovalent multispecific composition is administered to a patient to treat a neurological disease or disorder selected from brain cancer, a neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g., HIV-associated dementia). In additional embodiments, the multivalent and monovalent multispecific composition is administered to a patient to treat a brain cancer, metastatic cancer of the brain, or primary cancer of the brain. In additional embodiments, the multivalent and monovalent multispecific composition is administered to a patient to treat brain injury, stroke, spinal cord injury, or pain. Thus, according to some embodiments, the disease, disorder, or injury treated or prevented with an MRD-containing antibody or MRD of the invention is neurological. In one embodiment, the neurological disease, disorder or injury is associated with pain such as, acute pain or chronic pain.

In some embodiments the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4, or 5 targets associated with a neurological disease or disorder. In one embodiment, the multivalent and monovalent multispecific composition (e.g., MRD-containing antibody) binds 1, 2, or all 3 of the targets RGM A; NgR, and NogoA. In another embodiment, the multivalent and monovalent multispecific composition binds 1, 2, 3, or all 4 of RGM A, RGM B, and Semaphorin 3A or Semaphorin 4. In a further embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4 or 5 targets selected from aggrecan, midkine, neurocan, versican, phosphacan, Te38, TNF alpha, NogoA, RGM A, MAG, and OMGp. In another embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4 or 5 targets selected from aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF alpha. In an alternative embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4 or 5 targets selected from NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG and Omgp. In another embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4 or 5 targets selected from NGF, prostaglandin E2 (PGE2), TNF-alpha, IL1 beta, and IL6R.

In an additional embodiment, the multivalent and monovalent multispecific composition binds at least 1, 2, 3, 4 or 5 targets selected from alpha-synuclein, RGM A and one or more pro-inflammatory mediators (e.g., TNF alpha, IL1, and MCP-1). Such compositions have applications in, for example, treating neurodegenerative diseases such as, Parkinson's.

In another embodiment, the multivalent and monovalent multispecific composition binds and antagonizes (i.e., is an antagonist of e.g., inhibits the activity of) 1, 2, 3, 4 or 5 targets selected from RGM A, NOGO A, neurite inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4), ephrins and pro-inflammatory targets (e.g., IL12, TNFSF12 (TWEAK), IL23, CXCL13, TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), IL18, VEGF, VLA4, TNF alpha, CD45RB, CD200, interferon gamma, GMCSF, FGF, C5, CD52, and CCR2). In an additional embodiment, the multivalent and monovalent multispecific composition binds and antagonizes 1, 2, 3, 4 or 5 targets selected from CD3, IL2, IL2R, IL6, IL6R, IL10, IL12p40, IL23, TGF beta, TNFRSF21 (DR6), fn14, CD20, LINGO, CXCL13 and CCL2. The compositions have applications in treating for example, inflammation, neuroregeneration and neurodegenerative disorders, such as MS). Multivalent and multispecific compositions (e.g., MRD-containing antibodies) that bind at least 1, 2, 3, 4, 5 or more of these targets are also encompassed by the invention. In specific embodiments, the antibody component of the MRD-containing antibody binds CD3, CD20, CD52, VLA4, TNF, TNFRSF21 (DR6), LINGO, CD3, interferon gamma or IL6.

In another embodiment, the multivalent and monovalent multispecific composition binds and antagonizes (i.e., is an antagonist of) 1, 2, 3, 4 or 5 targets selected from AGE (S100 A, amphoterin), pro-inflammatory cytokines (e.g., IL1, IL6, and TNF), chemokines (e.g., MCP 1), and molecules that inhibit neural regeneration (e.g., Nogo and RGM A). These compositions have applications in treating, for example, chronic neurodegenerative diseases such as, Alzheimer's. In an additional embodiment, the composition of the invention binds at least 1, 2, 3, 4 or 5 targets that influence neural generation and survival including, for example, NGF agonists, IL1 or IL1R antagonists, and A-beta. These compositions have applications in treating, for example, chronic neurodegenerative diseases such as, Alzheimer's.

In an additional embodiment, the composition of the invention binds to and antagonizes 1, 2, 3, 4, or 5 targets that targets that interfere with neural regeneration or recovery, including NogoA, OMgp MAG, RGM A, CSPG, one or more astrocyte inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4), ephrins, and pro-inflammatory cytokines (e.g., IL1, IL6, and TNF). These compositions have applications in treating neurodegenerative diseases and neural injury or trauma.

In additional embodiment, the multivalent and monovalent multispecific composition binds and antagonize (i.e., is an antagonist of) 1, 2, 3, 4, or 5 targets associated with pain, including, but not limited to, NGF and SCN9A/NAV1.7. Such compositions have applications in for example, treating or alleviating pain and pain associated conditions.

In additional embodiments, the targets bound by the compositions of the invention binds and antagonizes 1, 2, 3, 4, 5 or more mediators and or soluble or cell surface targets implicated in the inhibition of neurite growth or recovery. In specific embodiments, compositions of the invention bind to and antagonizes 1, 2, 3, 4, 5 or more targets selected from Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines (e.g., IL1 and TNF alpha), chemokines (e.g., MIP 1a).

In some embodiments, the invention provides a method of treating or ameliorating pain by administering a therapeutically effective amount of a pain target binding MRD-antibody, to a patient in need thereof. In additional embodiments, the invention provides a method of treating or ameliorating pain by administering a therapeutically effective amount of an NGF binding MRD-antibody, to a patient in need thereof. In further embodiments, the invention provides a method of treating or ameliorating pain by administering a therapeutically effective amount of tanezumumab (e.g., Pfizer) comprising an MRD, to a patient in need thereof.

In additional embodiments, the invention provides a method of treating or ameliorating Alzheimer's by administering a therapeutically effective amount of an Alzheimer's target binding MRD-antibody, to a patient in need thereof. In additional embodiments, the invention provides a method of treating or ameliorating Alzheimer's by administering a therapeutically effective amount of a beta amyloid binding MRD-antibody, to a patient in need thereof. In additional embodiments, the invention provides a method of treating or ameliorating Alzheimer's by administering a therapeutically effective amount of RN1219 (PF-4,360,365; Pfizer) comprising an MRD, to a patient in need thereof.

In additional embodiments, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of a multiple sclerosis target binding MRD-antibody, to a patient in need thereof. In additional embodiments, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of a LINGO binding MRD-antibody, to a patient in need thereof. In another embodiment, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of an MRD-antibody that binds LINGO and TNFRSF21 (DR6) to a patient in need thereof. In additional embodiments, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of the Biogen LINGO antibody comprising an MRD, to a patient in need thereof. In further embodiments, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of the natalizumab (e.g., TYSABRI®; Biogen) comprising an MRD, to a patient in need thereof. In an additional embodiment, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of the Biogen LINGO antibody comprising an MRD, to a patient in need thereof.

In an additional embodiment, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of a CD20 binding MRD-antibody, to a patient in need thereof. In one embodiment, the invention provides a method of treating or ameliorating multiple sclerosis by administering a therapeutically effective amount of the ocrelizumab (Biogen Idec) comprising an MRD, to a patient in need thereof.

In other embodiments, the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are useful for treating or preventing an infectious disease. Infectious diseases that may be treated or prevented with multivalent and multispecific compositions (e.g., MRD-containing antibodies) include, but are not limited to, diseases associated with yeast, fungal, viral and bacterial infections. Viruses causing viral infections which can be treated or prevented with multivalent and multispecific compositions (e.g., MRD-containing antibodies) include, but are not limited to, retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), adenoviruses (e.g., lassa fever virus), paramyxoviruses (e.g., morbilbivirus virus, human respiratory syncytial virus, mumps, and pneumovirus), adrenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruscs (e.g., influenza viruses A, B and C (including avian influenza, e.g., H5N1 subtype)), papovaviruses (e.g., papillomaviruses), picomaviruses (e.g., rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses, reoviruses (e.g., rotaviruses), togaviruses (e.g., rubella virus), rhabdoviruses (e.g., rabies virus). Microbial pathogens causing bacterial infections include, but are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neissetia meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium pefringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigellaflexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia Lsutsugumushi, Chlamydia spp., and Helicobacter pylori.

In a preferred embodiment, the he multivalent and multispecific compositions (e.g., MRD-containing antibodies) are administered to a patient to treat or prevent human immunodeficiency virus (HIV) infection or AIDS, botulism, anthrax, or clostridium difficile.

VIII MRD Linked Compounds that are not Antibodies

In a distinct group of embodiments, one or more MRDs of the invention are operably linked to the amino and/or carboxy terminus of an immunoglobulin fragment, such as Fab, Fab′, F(ab′)2, pFc′, or Fc. In some embodiments, the MRDs are operably linked to a Fab or Fc polypeptide containing an additional Ig domain. In some embodiments, the MRDs are operably linked to the amino and/or carboxy terminus of an immunoglobulin fragment that is also operably linked to an scFv. In other embodiments, the MRDs of the invention are operably linked to an Fc-fusion protein.

According to this group of embodiments, one two, three, four, five, six, seven to ten, or more than ten MRDs are operably linked to the amino terminus and/or carboxy terminus of the immunoglobulin fragment. These MRDs are optionally linked to one another or to the immunoglobulin fragment via a linker. In one embodiment, one, two, three, four, five, six, seven to ten, or more than ten, of the MRDs operably linked to the amino terminus and/or carboxy terminus of the immunoglobulin fragment are the same. In another embodiment, one, two, three, four, five, six, seven to ten, or more than ten, of the MRDs operably linked to the amino terminus and/or carboxy terminus of the immunoglobulin fragment are different.

The MRDs operably linked to the immunoglobulin fragment can be monomeric (i.e., containing one MRD at the terminus of a peptide chain optionally connected by a linker) or multimeric (i.e., containing more than one MRD in tandem optionally connected by a linker). The MRDs can be homo-multimeric (i.e., containing more than one of the same MRD in tandem optionally connected by linker(s) (e.g., homodimers, homotrimers, homotetramers etc.)) or hetero-multimeric (i.e., containing two or more MRDs in which there are at least two different MRDs optionally connected by linker(s) where all or some of the MRDs linked to a particular terminus are different (e.g., heterodimer)). In one embodiment, two different monomeric MRDs are located at different termini of the immunoglobulin fragment. In another embodiment, three, four, five, six, or more different monomeric MRDs are located at different termini of the immunoglobulin fragment.

In an alternative embodiment, the MRD-containing antibody contains at least one dimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the MRD-containing antibody contains at least one homodimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the MRD-containing antibody contains at least one heterodimeric and one monomeric MRD located at different immunoglobulin termini.

In an alternative embodiment, the MRD-containing antibody contains at least one multimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the MRD-containing antibody contains at least one homomultimeric and one monomeric MRD located at different immunoglobulin termini. In another alternative embodiment, the MRD-containing antibody contains at least one heteromultimeric and one monomeric MRD located at different immunoglobulin termini.

Multiple MRDs that are operably linked to the immunoglobulin fragment can target the same target binding site, or two or more different target binding sites. Where the MRDs bind to different target binding sites, the binding sites may be on the same or different targets. Similarly, one or more of the MRDs may bind to the same target as the immunoglobulin fragment.

In some embodiments, at least one of the MRDs and if applicable, the immunoglobulin fragment (e.g., where the immunoglobulin fragment is an Fab), bind to their targets simultaneously. In additional embodiments, two, three, four, five, six, seven, eight, nine, ten, or more than ten MRDs, and if applicable the immunoglobulin fragment, bind to their targets simultaneously.

The synthesis of MRDs operably linked to an immunoglobulin fragment and the assay of these MRDs and immunoglobulin fragment for their ability to bind, or compete for binding with one or more targets simultaneously can be routinely accomplished using methods disclosed herein or otherwise known in the art.

In a specific embodiment, one or more of the operably linked MRDs or the immunoglobulin fragment, binds to VEGF. In another specific embodiment, one or more of the operably linked MRDs or the immunoglobulin fragment, binds to the same epitope as ranibizumab (LUCENTIS®, Genentech). In another specific embodiment, one or more of the operably linked MRDs or the immunoglobulin fragment, competitively inhibits ranibizumab binding to VEGF. In an additional embodiment, the immunoglobulin fragment is a Fab. In a further specific embodiment, the immunoglobulin fragment is ranibizumab.

In another embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of a VEGFA or VEGFR binding MRD-immunoglobulin fragment fusion to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of a VEGFA or VEGFR binding MRD-Fab fusion to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of MRD-ranibizumab to a patient in need thereof.

In other embodiments the one or more MRDs of the invention are operably linked to the amino and/or carboxyl terminus of an Fc fusion protein. The Fc fusion protein can contain fusions to any protein or polypeptide sequence of therapeutic value, for example, any of the targets or receptors of the targets described herein. For example, the fusions can contain the extracellular domain of receptors or ligands that typically function or display improved cognate-partner binding in multimeric form, including for example, receptors corresponding to the TNF-R superfamily (e.g., TNFR2, TACI, BCMA, HVEM, etc.), IL receptor superfamily (e.g., IL1-R-IL6R), VEGFR superfamily (e.g., VEGFR1-VEGR3), FGRFR superfamily (e.g., FGFR1-FGFR4), and B7 superfamily (e.g., CTLA)).

In a specific embodiment, one, two, three, four, five, six, or more MRDs are operably linked to a VEGR1/VEGFR2-Fc fusion protein. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as aflibercept (Regeneron). In another specific embodiment, one or more of the operably linked MRDs competitively inhibit aflibercept binding to VEGFA or PLGF. In a further specific embodiment, the MRDs are operably linked to aflibercept.

In another embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of an MRD-VEGFR1/VEGFR2-Fc fusion protein to a patient in need thereof. In a specific embodiment, the invention provides a method of treating colorectal cancer, prostate cancer, or non-small cell lung cancer comprising administering a therapeutically effective amount of a VEGFA or PLGF binding MRD-Fc fusion protein to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of a VEGFA or PLGF binding MRD-Fc fusion protein and irinotecan, 5FU, oxaliplatin, doxetaxel, or FOLFOX6, to a patient in need thereof.

In another embodiment, the invention provides a method of treating cancer comprising administering a therapeutically effective amount of MRD-aflibercept to a patient in need thereof. In a specific embodiment, the invention provides a method of treating colorectal cancer, prostate cancer, or non-small cell lung cancer comprising administering a therapeutically effective amount of MRD-aflibercept to a patient in need thereof. In a specific embodiment, the invention provides a method of treating macular degeneration comprising administering a therapeutically effective amount of MRD-aflibercept and irinotecan, 5FU, oxaliplatin, doxetaxel, or FOLFOX6, to a patient in need thereof.

In a specific embodiment, one, two, three, four, five, six, or more MRDs are operably linked to a CTLA4-Fc fusion protein. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as abatacept (ORENCIA®). In another specific embodiment, one or more of the operably linked MRDs competitively inhibits abatacept binding to CD80 (B7-1) or CD86 (B7-2). In a further specific embodiment, the MRDs are operably linked to abatacept. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as belatacept (Bristol Myers Squibb). In another specific embodiment, one or more of the operably linked MRDs competitively inhibits belatacept binding to CD80 (B7-1) or CD86 (B7-2). In an additional embodiment, the immunoglobulin fragment is a Fab. In a further specific embodiment, the MRDs are operably linked to belatacept.

In another embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of an MRD-CTLA4-Fc fusion protein to a patient in need thereof. In a specific embodiment, the invention provides a method suppressing an immune response comprising administering a therapeutically effective amount of MRD-abatacept to a patient in need thereof. In another specific embodiment, the invention provides a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of MRD-abatacept to a patient in need thereof. In another specific embodiment, the invention provides a method of suppressing an immune response to a graft rejection comprising administering a therapeutically effective amount of MRD-abatacept to a patient in need thereof.

In a specific embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of MRD-belatacept to a patient in need thereof. In another specific embodiment, the invention provides a method of suppressing an immune response to a graft rejection comprising administering a therapeutically effective amount of MRD-belatacept to a patient in need thereof.

In another specific embodiment, one, two, three, four, five, six, or more MRDs are operably linked to a TNFR2-Fc fusion protein. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as etanercept (ENBREL®). In another specific embodiment, one or more of the operably linked MRDs competitively inhibits etanercept binding to TNF alpha. In another embodiment, one or more of the operably linked MRDs binds ANG2. In a further specific embodiment, the MRDs are operably linked to etanercept.

In another embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of a MRD-TNFR2-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of a MRD-TNFR2-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of an MRD-TNFR2-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of an MRD-TNFR2-Fc fusion protein to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of an MRD-TNFR2-Fc fusion protein to a patient in need thereof. In another embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of an MRD-TNFR2-Fc fusion protein to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of an MRD-TNFR2-Fc fusion protein to a patient in need thereof.

In another embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of a MRD-etanercept-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof. In one embodiment, the invention provides a method of treating an inflammatory disorder, by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof. In another embodiment, the invention provides a method of treating Crohn's disease, by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof. In another embodiment, the invention provides a method of treating ulcerative colitis, by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof. In another embodiment, the invention provides a method of treating psoriatic arthritis, ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis by administering a therapeutically effective amount of MRD-etanercept to a patient in need thereof.

In another specific embodiment, one, two, three, four, five, six, or more MRDs are operably linked to a TACI-Fc fusion protein. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as atacicept (Merck/Serono). In another specific embodiment, one or more of the operably linked MRDs competitively inhibits atacicept binding to BLyS or APRIL. In a further specific embodiment, the MRDs are operably linked to atacicept.

In another embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of a MRD-TACI-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of a MRD-TACI-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of a MRD-TACI-Fc fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating systemic lupus erythematous by administering a therapeutically effective amount of a MRD-TACI-Fc fusion protein to a patient in need thereof. In another embodiment, the invention provides a method of suppressing an immune response comprising administering a therapeutically effective amount of an MRD-atacicept fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating an autoimmune disease by administering a therapeutically effective amount of an MRD-atacicept fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating rheumatoid arthritis, by administering a therapeutically effective amount of an MRD-atacicept protein fusion protein to a patient in need thereof. In one embodiment, the invention provides a method of treating systemic lupus erythematous, by administering a therapeutically effective amount of an MRD-atacicept fusion protein to a patient in need thereof.

In another specific embodiment, one, two, three, four, five, six, or more MRDs are operably linked to an IL1R-Fc fusion protein. In another specific embodiment, one or more of the operably linked MRDs bind to the same epitope as rilonacept (Regeneron). In another specific embodiment, one or more of the operably linked MRDs competitively inhibits rilonacept binding to IL1R. In a further specific embodiment, the MRDs are operably linked to rilonacept.

In another embodiment, the invention provides a method of preventing gout comprising administering a therapeutically effective amount of a MRD-IL1R-Fc fusion protein to a patient in need thereof. In a specific embodiment, the invention provides a method of preventing gout comprising administering a therapeutically effective amount of an MRD-rilonacept-Fc fusion protein to a patient in need thereof.

In some embodiments, the invention encompasses a complex comprising an antibody and at least one modular recognition domain (MRD), wherein the MRD comprises at least two cysteines, wherein a first cysteine is located within the first ten amino acids of the MRD, a second cysteine is located within the last ten amino acids of the MRD, and wherein the MRD comprises at least five amino acids between said first cysteine and said second cysteine. In additional embodiments, the MRD comprises at least 10, 15, 20, or 25 amino acids between the first cysteine and the second cysteine. In some embodiments, the MRD comprises at least one proline between the first cysteine and the second cysteine. In other embodiments, the MRD comprises at least two proline between the first cysteine and the second cysteine. In some embodiments, the first cysteine is no more than 5, 3, 3, 2, or 1 amino acids away from the N-terminus of the complex. In some embodiments, the second cysteine is no more than 5, 3, 3, 2, or 1 amino acids away from the C-terminus of the complex. In some embodiments, the in vivo half-life of an MRD in a complex of the invention is increased compared to the half-life of an MRD in a corresponding complex wherein at least one of the cysteines is mutated or deleted. In some embodiments, the binding affinity of the MRD is at least equal to the binding affinity of an MRD in a corresponding complex wherein at least one of the cysteines is mutated or deleted.

In some embodiments, the invention encompasses a complex comprising an antibody and at least one modular recognition domain (MRD), wherein the antibody and the MRD bind to different targets or epitopes on the same cell or molecule, wherein the MRD binding agonizes or antagonizes the MRD target under physiological conditions, and wherein said MRD does not bind to and agonize or antagonize said MRD target under physiological conditions in the absence of said antibody. In some embodiments, an MRD in the complex of the invention binds the MRD target in the absence of the antibody with an EC50 of greater than 0.01 nM, 0.1 nM, 0.5 nM, or 0.7 nM under physiological conditions.

In some embodiments, the invention encompasses a complex comprising an antibody and at least one MRD, wherein the antibody and the MRD bind to different targets or epitopes on a heteromeric or homomeric protein, wherein the MRD binding agonizes or antagonizes the MRD target under physiological conditions, and wherein said MRD does not bind to and agonize or antagonize said MRD target under physiological conditions in the absence of the antibody. In some embodiments, an MRD in the complex of the invention binds the MRD target with an EC50 of greater than 0.01 nM, 0.1 nM, 0.5 nM, or 0.7 nM under physiological conditions.

In some embodiments, the invention encompasses a method for inhibiting the growth of a cell comprising contacting the cell with a multispecific and multivalent complex comprising an antibody and at least one modular recognition domain (MRD), and a protein kinase inhibitor. In some embodiments, the antibody binds to a target selected from: VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and FGFR2. In some embodiments, the protein kinase inhibitor inhibits a target of the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits a different target than the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits more than one protein kinase. In some embodiments, the protein kinase inhibitor is a member selected from: imatinib, gefitinib, vandetanib, erlotinib, sunitinib, lapatinib, and sorafenib.

In some embodiments, the invention encompasses a method for inhibiting angiogenesis in a patient comprising administering to said patient a therapeutically effective amount of a multispecific and multivalent complex comprising an antibody and at least one modular recognition domain (MRD), and a protein kinase inhibitor. In some embodiments, the antibody binds to a target selected from: VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and FGFR2. In some embodiments, the protein kinase inhibitor inhibits a target of the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits a different target than the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits more than one protein kinase. In some embodiments, the protein kinase inhibitor is a member selected from: imatinib, gefitinib, vandetanib, erlotinib, sunitinib, lapatinib, and sorafenib.

In some embodiments, the invention encompasses a method for treating a patient having cancer comprising administering to said patient a therapeutically effective amount of a multispecific and multivalent complex comprising an antibody and at least one modular recognition domain (MRD), and a protein kinase inhibitor. In some embodiments, the antibody binds to a target selected from: VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and FGFR2. In some embodiments, the protein kinase inhibitor inhibits a target of the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits a different target than the MRD containing antibody. In some embodiments, the protein kinase inhibitor inhibits more than one protein kinase. In some embodiments, the protein kinase inhibitor is a member selected from: imatinib, gefitinib, vandetanib, erlotinib, sunitinib, lapatinib, and sorafenib.

In some embodiments, the invention encompasses a method for treating a patient having a disease or disorder of the immune system comprising administering to said patient a therapeutically effective amount of a multispecific and multivalent complex comprising an antibody and at least one modular recognition domain (MRD), and a protein kinase inhibitor. In some embodiments, the disease or disorder of the immune system is inflammation or an autoimmune disease. In further embodiments, the autoimmune disease is rheumatoid arthritis, Crohn's disease, systemic lupus erythematous, inflammatory bowel disease, psoriasis, diabetes, ulcerative colitis, or multiple sclerosis. In additional embodiments, the antibody binds TNF. In some embodiments, the protein kinase inhibitor inhibits a target that is not a target of the MRD-containing antibody. In some embodiments, the protein kinase inhibitor inhibits more than one protein kinase. In further embodiments, the protein kinase inhibitor is a member selected from: lestaurtinib, tofacitinib, ruxolitinib, SB1518, CYT387, LY3009104, TG101348, fostamatinib, BAY 61-3606, and sunitinib.

In some embodiments, the invention encompasses a multivalent and multispecific complex comprising an antibody and at least one modular recognition domain (MRD), wherein the complex has a single binding site for a cell surface target. In some embodiments, the multivalent and multispecific complex comprises 2 single binding sites for different epitopes on the same target. In some embodiments, the multivalent and multispecific complex has 2, 3, 4, 5 or more single binding sites for different targets. In some embodiments, the multivalent and multispecific complex has a single binding site for a target on a leukocyte. In some embodiments, the multivalent and multispecific complex has a single binding site for a target on a T-cell. In some embodiments, the multivalent and multispecific complex has a single binding site for CD3. In further embodiments, complex has a single binding site for CD3 epsilon. In additional embodiments, the complex has a single binding site for a target on a natural killer cell. In additional embodiments, the complex has multiple binding sites for a target on a diseased cell. In some embodiments, the complex has multiple binding sites for 2, 3, 4, 5 or more targets on a diseased cell. In additional embodiments, the complex has multiple binding sites for a target on a tumor cell. In further embodiments, the complex has multiple binding sites for 2, 3, 4, 5 or more targets on a tumor cell. In some embodiments, the complex has multiple binding sites for a target on an immune cell. In further embodiments, the complex has multiple binding sites for 2, 3, 4, 5 or more targets on an immune cell. In some embodiments, the complex has a single binding site for a target on a natural killer cell. In some embodiments, the complex binds a target on a leukocyte and a target on a tumor cell. In some embodiments, the complex binds CD3 and CD19. In further embodiments, the complex has multiple binding sites for a target on an infectious agent or a cell infected with an infectious agent. In further embodiments, the complex has multiple binding sites for 2, 3, 4, 5 or more targets on an infectious agent or a cell infected with an infectious agent. In some embodiments, the complex has a single binding site for a target associated with an endogenous blood brain barrier (BBB) receptor mediated transport system. In further embodiments, the complex has multiple binding sites for a target associated with an endogenous BBB receptor mediated transport system. In some embodiments, the complex has multiple binding sites for 2, 3, 4, 5 or more targets associated with an endogenous BBB receptor mediated transport system. In some embodiments, the single binding site is an MRD. In some embodiments, the single binding site is an antigen binding domain.

In some embodiments, the complexes of the invention comprise a cytotoxic agent.

Polynucleotide encoding a heavy chain or light chain of the MRD containing antibody of the invention, vectors comprising these polynucleotides and host cells containing these vectors and/or polynucleotides are also encompassed by the invention

The following examples are intended to illustrate but not limit the invention.

EXAMPLES Example 1. Integrin Targeting Antibody-MRD Molecules

Novel antibody-MRD fusion molecules were prepared by fusion of an integrin αvβ3-targeting peptides to catalytic antibody 38C2. Fusions at the N-termini and C-termini of the light chain and the C-termini of the heavy chain were most effective. Using flow cytometry, the antibody conjugates were shown to bind efficiently to integrin αvβ3-expressing human breast cancer cells. The antibody conjugates also retained the retro-aldol activity of their parental catalytic antibody 38C2, as measured by methodol and doxorubicin prodrug activation. This demonstrates that cell targeting and catalytic antibody capability can be efficiently combined for selective chemotherapy.

Example 2. Angiogenic Cytokine Targeting Antibody-MRD Molecules

Angiogenic cytokine targeting antibody-MRD fusion molecules were constructed. The antibody used was 38C2, which was fused with a MRD containing the 2×Con4 peptide (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEEC EWDPWTC EHMLE (SEQ ID NO:10)). The MRD-containing peptide was fused to either the N- or C-terminus of the light chain and the C-terminus of the heavy chain. Similar results were found with the other Ang2 MRD peptides. Additional Ang2 MRD peptides include: MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQT NFMPMDNDELLLY (SEQ ID NO:20) (LM-2x-32); and AQQEECEWDPWTCEHM GSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO:10) (2×Con4).

One of skill in the art, given the teachings herein, will appreciate that other such combinations will create functional Ang2 binding MRDs as described herein.

Example 3. Antibody-MRD Fusions with Non-Catalytic Antibodies

A humanized mouse monoclonal antibody, LM609, directed towards human integrin αvβ3 has been previously described (Rader, C. et. al., PNAS 95:8910-5 (1998)).

A human non-catalytic monoclonal Ab, JC7U was fused to an anti-Ang2 MRD containing 2×Con4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEEC EWDPWTCEH MLE (SEQ ID NO:10)) at either the N- or C-terminus of the light chain. 2×Con4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWT CEHMLE (SEQ ID NO:10)) was studied as an N-terminal fusion to the Kappa chain of the antibody (2×Con4-JC7U) and as a C-terminal fusion (JC7U-2×Con4). Both fusions maintained integrin and Ang2 binding. As shown in the left panel of FIG. 3, both antibody constructs (2×Con4-JC7U and JC7U-2×Con4) specifically bound to recombinant Ang2 as demonstrated by ELISA studies. Binding to Ang2, however, is significantly higher with JC7U-2×Con4, which has the 2×Con4 (SEQ ID NO:10) fusion at the C-terminus of the light chain of the antibody. The right panel of FIG. 3 depicts the binding of Ang2-JC7U and JC7U-Ang2 to integrin αvβ3. The results show that fusion of 2×Con4 (SEQ ID NO:10) to either the N- or the C-light chain terminus does not affect mAb JC7U binding to integrin αvβ3. FIG. 4 depicts another ELISA study using the same antibody-MRD fusion constructs.

Example 4. HERCEPTIN®-MRD Fusion Molecules

Another example of MRD fusions to a non-catalytic antibody are HERCEPTIN®-MRD fusion constructs. The HERCEPTIN®-MRD fusions are multifunctional, both small molecule αv integrin antagonists and the chemically programmed integrin-targeting antibody show remarkable efficacy in preventing the breast cancer metastasis by interfering with αv-mediated cell adhesion and proliferation. MRD fusions containing HERCEPTIN®-2×Con4 (which targets ErbB2 and Ang2) and HERCEPTIN®-V114 (which targets ErbB2 and VEGF targeting) and HERCEPTIN®-RGD-4C-2×Con4 (which targets ErbB2, ang2, and integrin targeting) are effective.

Example 5. VEGF Targeting Antibody-MRD Molecules

An antibody containing an MRD that targets VEGF was constructed. A MRD which targets vl 14 (SEQ ID NO:13) was fused at the N-terminus of the kappa chain of 38C2 and HERCEPTIN® using a linker. Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong VEGF binding.

Example 6. IGF1R Targeting Antibody-MRD Molecules

Fusion of an MRD which targets IGF1R (SFYSCLESLVNGPAEKSRG QWDGCRKK (SEQ ID NO:14)) to the N-terminus of the kappa chain of 38C2 and HERCEPTIN® using the long linker sequence as a connector was studied. Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong IGF1R binding. Additional clones showing high binding to IGR1R were identified after several rounds of mutagenesis and screening of the regions described in Table 4. The preferred sequences listed in Table 5 bind IGF1R and show no significant or no binding affinity to the insulin receptor, thereby suggesting specificity for IGF1R.

TABLE 4 Template for further mutagenesis. Name DNA AA Rm2-2-218 GTGGAGTGCAGGGCGCCG VECRAP (SEQ ID NO: 51) (SEQ ID NO: 50) Rm2-2-316 GCTGAGTGCAGGGCTGGG AECRAG (SEQ ID NO: 53) (SEQ ID NO: 52) Rm2-2-319 CAGGAGTGCAGGACGGGG QECRTG (SEQ ID NO: 55) (SEQ ID NO: 54)

TABLE 5 SEQ ID Mutant Amino acid sequence Template NO Rm4-31 NFYQCIEMLASHPAEKSRGQWQECRTGG Rm2-2-319 35 Rm4-33 NFYQCIEQLALRPAEKSRGQWQECRTGG Rm2-2-319 36 Rm4-39 NFYQCIDLLMAYPAEKSRGQWQECRTGG Rm2-2-319 37 Rm4-310 NFYQCIERLVTGPAEKSRGQWQECRTGG Rm2-2-319 38 Rm4-314 NFYQCIEYLAMKPAEKSRGQWQECRTGG Rm2-2-319 39 Rm4-316 NFYQCIEALQSRPAEKSRGQWQECRTGG Rm2-2-319 40 Rm4-319 NFYQCIEALSRSPAEKSRGQWQECRTGG Rm2-2-319 41 Rm4-44 NFYQCIEHLSGSPAEKSRGQWQECRTG Rm2-2-319 42 Rm4-45 NFYQCIESLAGGPAEKSRGQWQECRTG Rm2-2-319 43 Rm4-46 NFYQCIEALVGVPAEKSRGQWQECRTG Rm2-2-319 44 Rm4-49 NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45 Rm4-410 NFYQCIEVFWGRPAEKSRGQWQECRTG Rm2-2-319 46 Rm4-411 NFYQCIEQLSSGPAEKSRGQWQECRTG Rm2-2-319 47 Rm4-415 NFYQCIELLSARPAEKSRGQ WAECRAG Rm2-2-316 48 Rm4-417 NFYQCIEALARTPAEKSRGQWVECRAP Rm2-2-218 49

Example 7. ErbB2 Binding, Ang2-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets Ang2 (L17) (SEQ ID NO:7) fused to the light chain of an antibody which binds to ErbB2. Either the short linker sequence, the long linker sequence, or the 4th loop in the light chain constant region was used as a linker. FIG. 5 depicts the results of an ELISA using constructs containing an N-terminal fusion of an Ang2 targeting MRD with the ErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO:1)) (L17-sL-Her), a C-terminal fusion of Ang2 targeting MRD with the ErbB2 antibody with the short linker peptide (Her-sL-L17), a C-terminal fusion of Ang2 targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-L17), or an N-terminal fusion of Ang2 targeting MRD with the ErbB2 antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO:19)) (L17-1L-Her). ErbB2 was bound with varying degrees by all of the constructs. However, Ang2 was bound only by Her-sL-L17 and L17-1L-Her.

Example 8. Hepatocyte Growth Factor Receptor Binding, Ang2-Targeting Antibody-MRD Molecules

Fusion of an MRD which targets Ang2 (L17) (SEQ ID NO:7) was made to either the N-terminus or C-terminus of the light chain of the Met antibody, which binds to hepatocyte growth factor receptor. Either the short linker sequence or the long linker sequence were used as a connector. FIG. 6 depicts the results of an ELISA using constructs containing N-terminal fusion of Ang2 targeting MRD with the Met antibody with the short linker peptide (GGGS (SEQ ID NO:1)) (L17-sL-Met), N-terminal fusion of Ang2 targeting MRD with the Met antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO:19)) (L17-1L-Met), and C-terminal fusion of Ang2 targeting MRD with the Met antibody with the long linker peptide (Met-ILL17). Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong Ang2 binding when the long linker peptide was used. Fusion of the Ang2 targeting MRD to the C-light chain terminus of the antibody resulted in slightly higher binding to Ang2 then fusion of the Ang2 targeting to the N-light chain terminus of the antibody.

Example 9. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C) with the sequence CDCRGDCFC (SEQ ID NO:106) fused to the light chain of an antibody HERCEPTIN® which binds to ErbB2 (Her). Either the short linker sequence, the long linker sequence, or the 4th loop in the light chain constant region was used as a linker. FIG. 7 depicts the results of an ELISA using constructs containing an N-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO:1)) (RGD4C-sL-Her), a C-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the short linker peptide (Her-sL-RGD4C), a C-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-RGD4C), or an N-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO:19)) (RGD4C-1L-Her). ErbB2 was bound with varying degrees by all of the constructs. However, integrin αvβ3 was bound only by RGD4C-1L-Her.

Example 10. Hepatocyte Growth Factor Receptor Binding, Integrin-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C) (SEQ ID NO:106) fused to the light chain of an antibody which binds to the hepatocyte growth factor receptor (Met). Antibody-MRD constructs containing the long linker sequence were used. FIG. 8 depicts the results of an ELISA using constructs containing an N-terminal fusion of integrin αvβ3 targeting MRD with the hepatocyte growth factor receptor antibody (RGD4C-1L-Met), or a C-terminal fusion of integrin αvβ3 targeting MRD with the hepatocyte growth factor receptor antibody (Met-1L-RGD4C). The RGD4C-1L-Met demonstrated strong integrin αvβ3 binding.

Example 11. ErbB2 Binding, Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules

Antibodies were constructed which contains an MRD that targets insulin-like growth factor-I receptor (RP) (SEQ ID NO:14) fused to the light chain of an antibody which binds to ErbB2 (Her). Either the short linker peptide, the long linker peptide, or the 4th loop in the light chain constant region was used as a linker (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992); U.S. Pat. No. 5,677,171; and ATCC Deposit 10463, each of which is herein incorporated by reference). FIG. 9 depicts the results of an ELISA using constructs containing an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the short linker peptide (RP-sL-Her), a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody and the short linker peptide (Her-sL-RP), a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-RP), an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the long linker peptide (RP-1L-Her), or a C-terminal fusion of insulin-like growth factor-1 receptor targeting MRD with the ErbB2 antibody with the long linker peptide (Her-1L-RP). ErbB2 was bound with varying degrees by all of the constructs. Insulin-like growth factor-I receptor was bound by RP-1L-Her.

Example 12. ErbB2 Binding. VEGF-Targeting Antibody-MRD Molecules

Fusion of an MRD which targets VEGF (Vl 14) (SEQ ID NO:13) (Fairbrother W. J., et al., Biochemistry 37:177754-177764 (1998)) was made to the N-terminus of the light chain of a ErbB2-binding antibody (Her). A medium linker peptide (SSGGGGSGGGGGGSS (SEQ ID NO:2)) was used as a connector. FIG. 10 depicts the results of an ELISA using a construct containing an N-terminal fusion of VEGF targeting MRD with the ErbB2-binding antibody with the medium linker peptide (Vl 14-mL-Her). Expression and testing of the resulting antibody-MRD fusion construct demonstrated strong VEGF and ErbB2 binding.

Example 13. Integrin Targeting Antibody-MRD Molecules

Fusion of an MRD which targets integrin αvβ3 (RGD) (SEQ ID NO:106) to the N-terminus of the light chain of 38C2 using the medium linker peptide as a connector was studied. FIG. 11 demonstrates that expression and testing of the resulting antibody-MRD fusion construct had strong integrin αvβ3 binding.

Example 14. Ang2 Targeting Antibody-MRD Molecules

Fusion of an MRD which targets Ang2 (L 17) (SEQ ID NO:7) to the C-terminus of the light chain of 38C2 using the short linker sequence as a connector was studied. FIG. 12 demonstrates that expression and testing of the resulting antibody-MRD fusion construct had strong Ang2 binding.

Example 15. ErbB2 Binding, Integrin and Ang2 Targeting Antibody-MRD Molecules

An MRD which targets integrin αvβ3 (RGD4C) was connected to the N-terminus of the light chain of an ErbB2 targeting antibody (Her) with a medium linker, and an Ang2 (L17) targeting MRD was connected by a short linker to the C-terminus of the same ErbB2 targeting antibody (RGD4C-mL-Her-sL-L17). FIG. 13 demonstrates that the resulting antibody-MRD fusion construct bound to integrin, Ang2, and ErbB2.

Similarly, ErbB2 targeting antibodies (e.g., Her) with an IGF-1R MRD fused to the C-terminus of the heavy chain or the N-terminus of the light chain bound to immobilized IGF-1R at comparable rates. In addition, ErbB2 targeting antibodies containing an IGF-1R MRD fused to the N-terminus of the light chain and an Ang2 MRD fused to the C-terminus of the heavy chain bound to immobilized IGF-1R at comparable rates. Each of these three multivalent and multispecific compositions (e.g., MRD-containing antibodies) also inhibited the binding of IGF-1 to immobilized IGF-IR. The trispecific molecule (HERCEPTIN with 1GF-1R and Ang2 MRDs) bound to both cell surface ErbB2 and soluble Ang2.

Example 16. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C) fused to the N-terminus of the heavy chain of an antibody which binds to ErbB2 (Her) using the medium linker as a connector (RGD4C-mL-her-heavy). FIG. 14 depicts the results of an ELISA using the construct. Both integrin and ErbB2 were bound by the construct.

Example 17. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin, Ang2 or Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules with the Short Linker Peptide

Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth factor receptor binding antibodies, and integrin αvβ3, Ang2 or insulin-like growth factor-I receptor-targeting MRD regions were linked with the short linker peptide to the light chain of the antibody. FIG. 15 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang2 targeting MRD fused to the ErbB2 antibody (L17-sL-Her), an N-terminal fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-sL-Her), an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2-binding antibody (RP-sL-Her), a C-terminal fusion of Ang2 targeting MRD with the hepatocyte growth factor receptor binding antibody (L17-sL-Met), a C-terminal fusion of Ang2 targeting MRD with the ErbB2-binding antibody (Her-sL-L17), a C-terminal fusion of integrin targeting MRD with the ErbB2-binding antibody (Her-sL-RGD4C), or a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2-binding antibody (Her-sL-RP). ErbB2 was bound with varying degrees by the antibody-MRD constructs, with the exception of the construct containing the hepatocyte growth factor receptor-binding antibody. Antigen was bound only by the Her-sL-L17 construct.

Example 18. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin, Ang2 or Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules with the Long Linker Peptide

Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth factor receptor binding antibodies, and integrin αvβ3, Ang2 or insulin-like growth factor-I receptor-targeting MRD regions linked with the long linker peptide to the light chain of the antibody. FIG. 16 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang2 targeting MRD fused to the ErbB2 antibody (L17-1L-Her), an N-terminal fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-1L-Her), an N-terminal fusion of insulin-like growth factor-I receptor-targeting MRD with the ErbB2-binding antibody (RP-1L-Her), a C-terminal fusion of Ang2 targeting MRD with the hepatocyte growth factor receptor binding antibody (L17-1L-Met), a C-terminal fusion of integrin targeting MRD with the hepatocyte growth factor receptor binding antibody (RGD4C-1L-Met), a C-terminal fusion of Ang2 targeting MRD with the insulin-like growth factor-I receptor binding antibody (Her-1L-RP), a C-terminal fusion of Ang2 targeting MRD with the hepatocyte growth factor receptor binding antibody (Met-1L-L17), or a C-terminal fusion of integrin targeting MRD with the hepatocyte growth factor receptor binding antibody (Met-1L-RGD4C). As shown in FIG. 16, antibody-MRD fusions are effective to bind antigen and ErbB2. Lu et al., J. Biol. Chem. 20:280(20):19665-19672 (2005); Lu et al., J. Biol. Chem.: 279(4):2856-65 (2004).

Example 19. Cloning and Expression of Ang2 MRDs Fused to Maltose Binding Protein

A. Cloning of MBP Fusions

Monomer and dimer peptides were expressed as protein fusions to maltose binding protein (MBP) using a modified form of the pMAL-p2 vector and expression system from New England Biolabs (NEB; Beverly, Mass.) The PCR-generated MRD sequence was inserted into a pMAL vector down-stream from the malE gene, which encodes MBP. This results in a vector that encodes an MRD-MBP-fusion protein. The pMAL vector contains a strong Ptac promoter and is inducible by IPTG. The pMAL-p2 series contains the normal malE signal sequence, which directs the fusion protein through the cytoplasmic membrane. pMAL-p2 fusion proteins capable of being exported can be purified from the periplasm through osmotic shock. Further purification can be performed, for example by binding to amylose resin.

B. Expression of MBP Fusion Proteins and Osmotic Shock Fractionation

For expression of fusion proteins, bacterial cultures grown overnight were back-diluted into fresh media to an OD A600 of approximately 0.1. Cultures were grown to an OD of approximately 0.8 and induced with IPTG at a concentration of 0.3 mM. Cultures were incubated with shaking for approximately 4 hours, after which bacteria were centrifuged for 15 minutes at 4700 g. Pelleted bacteria were resuspended in 30 mM Tris-HCL pH 7.4, 20% sucrose, 1 mM EDTA. Cells were incubated for 20 minutes at room temperature (RT) prior to centrifugation for 15 minutes at 4700 g. Pelleted bacteria were then resuspended in ice cold MgSO₄, and incubated for 20 minutes on ice, with periodic mixing. Cell suspensions were sonicated (Misonix XL2020) for 90 seconds. Cells were centrifuged at 4° C. for 20 minutes at 4700 g. The supernatant (“osmotic shock fraction”) was adjusted to 1×PBS using 10×PBS (Quality Biologics, cat #119-069-131) and filtered through 0.2 micron filter. These osmotic shock fractions were assayed directly for binding to Ang2.

C. Direct Binding of MBP Fusion Proteins

For detection of direct binding of MRD-MBP fusions to Ang2, the following ELISA was performed. Ninety-six-well plates were coated overnight with rhAng2 (R&D cat#623-AN) at 320 ng/ml (100W/well). Wells were blocked for 3.25 hours with 250 μl Blocking buffer (Thermo Cat# N502), followed by 4 washes with 300 μl wash buffer (PBS, 0.1% tween). MBP fusion proteins were serially diluted in Blocking buffer and added to wells for 2 hours at RT. After washing (8×300 μl wash buffer), samples were treated with HRP-mouse anti MBP mAb (NEB, cat # E8038S), diluted 1:4000 in Blocking buffer. After incubation for 1 hour at RT, wells were washed (8×300 μl wash buffer) prior to receiving 100 μl of TMB substrate (KPL Laboratories). Color development was stopped with 100 μl of H₂SO₄, and absorbance was read at 450 nm.

D. Results

MRD-MBP fusions were assayed for direct binding to Ang2. Osmotic shock fractions of induced bacterial cultures were serially diluted and added to Ang2 coated wells. Bound fusion proteins were detected with anti-MBP mAb. The dose response curves are presented in FIG. 17A. Assayed proteins represent mutational variants of the sequence MGAQTNFMPMDDDE LLLYEQFILQQGLE (L17D) (SEQ ID NO:107). In this series, the motif MDD within L17D was mutated at the first D to all other possible amino acids (except cysteine). Other MRDs tested were “Lm32 KtoS” and a dimer of Lm32 (2×Lm32). As presented in FIG. 17B, several MXD mutants exhibit binding in the 0.1 to 100 nm range. The Lm32 dimer (2×Lm32) exhibits greater than 10 fold higher affinity for Ang2 than either L17D or “Lm32 KtoS”.

Example 21. Expression and Purification of Antibodies Containing MRDs

Molecular recognition domains were constructed and expressed in a pcDNA 3.3 vector as fusion proteins with either the heavy or light chains of antibodies. For protein production, plasmid DNAs encoding the heavy and light chains of the antibodies containing MRDs were first transformed into chemically competent bacteria in order to produce large amounts of DNA for transient transfection. Single transformants were propagated in LB media and purified using Qiagen's Endotoxin Free Plasmid Kits. Briefly, cells from an overnight culture were lysed; lysates were clarified and applied to an anion-exchange column, and then subjected to a wash step and eluted with high salt. Plasmids were precipitated, washed, and resuspended in sterile water.

HEK293T cells were expanded to the desired final batch size (about 5 L) prior to transfection. The purified plasmid (1 mg per liter of production) was complexed with the polyethylenimine (PEI) transfection reagent, added to the shake flask culture, and incubated at 37° C. The culture was monitored daily for cell count, cell diameter, and viability. The conditioned medium was harvested and stored at −80° C. until purification.

Antibodies containing MRDs were purified from the conditioned medium using affinity chromatography. Culture supernatant was filter clarified and applied directly to a chromatography column containing recombinant Protein A Sepharose (GE Healthcare). The column was washed, and bound antibodies containing MRDs were eluted by lowering buffer pH. Following elution, eluate fractions were immediately adjusted to physiologic pH. Following Protein A affinity purification, an additional optional polishing chromatographic step can be performed as needed.

Purified proteins were dialyzed into PBS, concentrated to ˜1-4 mg/ml, sterile filtered, aliquoted aseptically, and stored frozen at −80° C. All steps of the purification were monitored by SDS-PAGE-Coomassie, and precautions were taken during the purification to keep endotoxin levels as minimal as possible.

The final product was analyzed for endotoxin levels (EndoSafe), purity (SDS-PAGE-coomassie, analytical SEC-HPLC), protein identity (Western blot), and yield (Bradford assay). An additional size exclusion HPLC analysis was performed to assess the level of aggregates.

The data presented in Table 6 indicate that the antibodies containing MRDs can be expressed and purified using conventional techniques.

TABLE 6 Zybody Yield (mg) Purity Aggregates (%) Endotoxin (EU/ml) HER2xCon4(H) 36 >90% 4.6 <1 HER-lm32(H) 57 >90% 1 2.02 HER-lm32(L) 98 >90% 2 3.26 AVA-lm32(H) 12 >90% 0 <1

Example 22. Simultaneous Binding of HER Lm32(H) and HER Lm32 (L) to Her2 and Ang2

A. Methods

Ninety-six-well plates were coated overnight with rHER2-Fc (R&D cat#1129-ER-050) at 20 ng/ml (100 μl/well). Wells are blocked for 3.25 hours with 250 μl Blocking buffer (Thermo Cat# N502), followed by 4 washes with 300 μl wash buffer (PBS, 0.1% tween). Antibodies containing MRDs (HER-lm32(H), HER-lm32(L), and AVA-lm32(H)) and antibodies (HERCEPTIN®) were serially diluted in Blocking buffer, containing 1.94 μg/ml biotinylated Ang2 (R&D cat#BT633) and added to wells for 2 hours at RT. After washing (8×300 μl wash buffer), parallel samples received either HRP-conjugated anti-human kappa chain mAb-(Abcam, cat # ab79115-1) diluted 1:1000 in Blocking buffer or HRP-conjugated streptavidin (Thermo Scientific cat#N100) diluted 1:4000 diluted in Blocking buffer. After incubation for 1 hour at RT, wells were washed (8×300 μl wash buffer) prior to receiving 100 μl of TMB substrate (KPL Laboratories). Color development was stopped with 100 μl of H₂SO₄, and absorbance was read at 450 nm.

B. Results

As detected with anti-human kappa chain mAb, both a HERCEPTIN®-based antibody or HERCEPTIN®-based antibodies containing MRDs bind to Her2 Fc in the presence of Ang2 in a dose dependent manner (FIG. 18A). Only the HERCEPTIN®-based antibodies containing MRDs (HER-lm32(H) and HER-lm32(L)) exhibit simultaneous binding to Her2 Fc and Ang2, as detected by HRP-conjugated streptavidin (FIG. 18B).

Example 23. Simultaneous Binding of AVA-lm32(H) to VEGF and Ang2

A. Methods:

Ninety-six-well plates were coated overnight with human VEGF (PeproTech, Inc. cat#100-20) at 30 ng/ml (100 μl/well). Wells were blocked for 3.25 hours with 250 μl Blocking buffer (Thermo Cat# N502), followed by 4 washes with 300 μl wash buffer (PBS, 0.1% tween). Antibodies containing MRDs (HER-lm32(H) and AVA-lm32(H)) and antibodies (AVASTIN®) were serially diluted in Blocking buffer, containing 3.876 μg/ml biotinylated Ang2 (R&D cat#BT633) and added to wells for 2 hours at RT. After washing (8×300 μl wash buffer), parallel samples received either HRP-conjugated anti-human kappa chain mAb (Abcam, cat # ab79115) diluted 1:1000 in Blocking buffer or HRP-conjugated streptavidin (Thermo Scientific cat#N100) diluted 1:4000 diluted in Blocking buffer. After incubation for 1 hour at RT, wells were washed (8×300 μl wash buffer) prior to receiving 100 of TMB substrate (KPL Laboratories). Color development was stopped with 100 μl of H₂SO₄, and absorbance was read at 450 nm.

B. Results

As detected with anti-human kappa chain mAb, both AVASTIN® and AVASTIN®-based antibodies containing MRDs bind to VEGF in the presence of Ang2 in a dose dependent manner (FIG. 19A). Only the AVASTIN®-based antibodies containing MRDs (AVA-lm32(H)) exhibited simultaneous binding to VEGF and Ang2, as detected by HRP-conjugated streptavidin (FIG. 19B).

Example 24. Simultaneous Binding of HER-lm32 (H) and HER-lm32 (L) to HER2 and Angiopoietin-2

The ability of HER-lm32 (H) and HER-lm32 (L) simultaneously bind to Her2 expressed on the surface of breast carcinoma cells BT-474, and to Ang2 in solution, was determined by flow cytometry. Mouse anti-human Ig-FITC was used for detection of the heavy chain of the antibodies containing MRDs, and Ang2-biotin/streptavidin-PE was used for detection of the lm32 MRD. Cells that bind Her2 and Ang2 simultaneously are expected to be detected as double positive for FITC and PE fluorescence.

One million HER2 positive breast carcinoma cells BT-474 were incubated with 1 μg HER-lm32(H) or HER-lm32(L) for 25 minutes at RT. After washing, cells were incubated with 200 ng/mL Ang2 biotin (R&D systems) for 25 minutes at RT and then with 20 μL of mouse anti-human Ig-FITC and Streptavidin-PE for 15 minutes. After washing with 2 mL buffer, cells were analyzed by flow cytometry (FACS Canto 11, BD).

In order to confirm the specificity of binding of HER-lm32(H) and HER-lm32(L) to HER2 on BT-474 cells, binding was determined in the presence of 10-fold excess of HERCEPTIN®. In these experiments, antibodies containing MRDs (1 μg) were incubated with one million BT-474 cells in the absence or presence of 10 μg HERCEPTIN® for 25 minutes at RT. Binding of antibodies containing MRDs to HER2 was determined by incubating with 200 ng/mL Ang2 biotin followed by detection with streptavidin-PE.

The data presented in FIG. 20A demonstrate that both HER-lm32(H) and HER-lm32(L), bind simultaneously to HER2 and Ang2. In both cases, the cells exhibited bright dual fluorescence in the FITC and PE fluorescence channels. The fact that HER-lm32(H) and HER-lm32(L) binding to HER2 is completely inhibited by HERCEPTIN® (FIG. 20B) indicates that the binding is specific.

In addition, tri-specific binding was demonstrated using an antibody containing two distinct MRDs. An EGFR-binding affibody and an Ang2-binding peptide (LM32) were fused to the C-terminus of the light and heavy chains of HERCEPTIN, respectively. The MRD-containing antibody was incubated with the EGFR+, Her2-A431 human epithelial cell line. Cell-bound MRD-containing antibody was detected with biotinylated Ang2/strepavidin-PE, alexafluor labeled ErbB2-Fc, or the combination of Ang2/strepavidin-PE and alexafluor labeled ErbB2-Fc. The results demonstrated that the MRD-containing antibody simultaneously bound EGFR cell surface receptor and two soluble ligands (ErbB2 and Ang2).

Additional experiments demonstrated that a fusion of lm32 to the C-terminus of the heavy chain of HERCEPTIN retained the binding specificity and Fc function of HERCEPTIN. HERCEPTIN and the HERCEPTIN-lm32 fusion bound to FcRn with similar affinities (EC50s for HERCEPTIN and HERCEPTIN lm-32 were 2.17 and 2.84 μg/ml, respectively). The HERCEPTIN and the HERCEPTIN-lm32 fusion displayed comparable ADCC activity on SK-BR-3 cells. The HERCEPTIN-lm32 fusion and HERCEPTIN bound to Fcγ-RI and Fcγ-RIII with similar affinities. The HERCEPTIN-lm32 fusion and HERCEPTIN bound to complement receptor C1q with similar affinities. In addition, the HERCEPTIN-lm32 fusion bound Ang2 with subnanomolar affinity and antagonized Ang2 binding to the Tie-2 receptor. The HERCEPTIN-lm32 fusion bound the extracellular domain of ErbB2 and also inhibited Ang2-induced proliferation of primary bovine lymphoendothelial cells. The HERCEPTIN-lm32 fusion and HERCEPTIN bound to the extracellular domain of Her2 with similar kinetic parameters. Additional experiments demonstrated that the HERCEPTIN-lm32 fusion was as effective as HERCEPTIN in inhibiting the proliferation of several cultured breast cancer cell lines (BT-474, MDA-MB-361 and SK-BR-3). The anti-proliferative effect of the HERCEPTIN-lm32 fusion on SK-BR-3 cells was not affected by the presence of Ang2 (2 μg/ml) in the culture.

Simultaneous target binding has been observed for other multivalent and multispecific compositions (e.g., multivalent and multispecific compositions (e.g., MRD-containing antibodies)). For example, a fusion of lm32 to the C-terminus of HUMIRA heavy chain (HUM-lm32(H)) was able to simultaneously bind to Ang2 and TNFα. The same fusion was able to bind 293 cells transiently transfected with full length human TNFα with similar affinity to the HUMIRA antibody. HUM-lm32(H) was also able to inhibit the interaction of TNF with its receptors. HUM-lm32(H) also bound to cell surface expressed and plate bound FcRn as well as to Fcγ-RI and Fcγ-RIII.

Example 25. Antibody-MRDs Containing Heavy Chain Fusions Bind to Targets

To assess the ability of lm32-containing antibodies to block the interaction of Ang2 with its receptor TIE2, their effect on the binding of soluble TIE2 to plate-bound Ang2 was determined by ELISA.

Ang2 (R&D Systems, catalog#623-AN) was coated on a 96-well plate (Thermo Electron, cat#3855) at 200 ng/mL in PBS overnight at 4° C. The plate was then incubated with 100 μl, of blocking solution (Thermo Scientific, cat#N502) for 1 hour at RT. After washing the plate 4 times with 0.1% Tween-20 in PBS, the plate bound Ang2 was incubated with 0.5 μg/mL soluble TIE2 (R&D Systems, cat#313-TI,) in the absence or presence of various concentrations of serially diluted antibodies containing MRDs for 1 hour at RT. After washing 4 times, 100 μL of 0.5 μg/mL anti TIE2 antibody (cat#BAM3313, R&D Systems) was added and incubated at RT for 1 hour. TIE2 binding to Ang2 was detected by incubation with 1:1000 diluted goat anti-mouse-HRP (BD Pharmingen, cat#554002) for 1 hour at RT. The plate was washed 4 times and incubated with 100 μL TMB reagent for 10 minutes at RT. After stopping the reaction with 100 μL of 0.36N H₂SO₄, the plate was read at 450 nm using a spectrophotometer.

As presented in FIG. 21A, HER-lm32(H), HER-lm32(L), and AVA-lm32(H) inhibited TIE2 binding to plate-bound Ang2 in a dose-dependent fashion. All tested lm32-containing antibodies demonstrated comparable inhibitory effects with IC-50 values of 4 nM for HER-lm32 (H), 8 nM for HER-lm32(L) and 3.3 nM for AVA-lm32(H).

Example 26. Antibody-MRDs Containing Heavy Chain Fusions Bind to Targets

To determine the specificity and relative affinity of AVA-lm32 (H) binding to VEGF, a competitive binding assay was performed using biotin labeled AVASTIN®.

AVASTIN® was labeled with biotin using EZ-Link NHS-LC-Biotin (Pierce, cat#21336). VEGF (Peprotech, cat#100-20) was coated on a 96-well plate (Thermo Electron, cat#3855) at 100 ng/mL in PBS overnight at 4° C. The plate was then incubated with 100 μL of blocking solution (Thermo Scientific, cat#N502) for 1 hour at RT. After washing the plate 4 times with 0.1% Tween 20 in PBS, 50 μL of AVASTIN®-biotin at 150 ng/mL and 50 μL of various concentrations of AVA-lm32(H) or unlabeled AVASTIN® were added and incubated at RT for 1 hour. The plate was washed 4 times and incubated with Streptavidin-HRP (Thermo, cat#N100) at 1:1000 dilution for 1 hour at RT. The plate was washed 4 times and 100 μL of TMB reagent was added. After 10 minutes incubation at RT, 100 μL of 0.36N H₂SO₄ was added to stop the reaction and the plate was read at 450 nm.

The data presented in FIG. 22 demonstrate that AVA-lm32(H) specifically binds to VEGF-2. It inhibits binding of biotinylated AVASTIN® to VEGF in a dose dependent manner. The dose response curves generated by AVA-lm32(H) and unlabeled AVASTIN® are superimposable and indicate similar binding affinities.

Example 27. Binding of HER-lm32(H) and HER-lm32(L) to HER2 Expressed on Breast Cancer Cells

To determine the relative binding affinity of HERCEPTIN®-based antibodies containing MRDs to cell surface HER2 compared to HERCEPTIN®, a competitive binding assay was performed with Eu-labeled HERCEPTIN®.

HERCEPTIN® was labeled with Eu3+ using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) Europium-labeling kit (Perkin Elmer Life Sciences, cat#1244-302) following the manufacturer's instructions. The labeling agent is the Eu-chelate of N1-(p-isothiocynateobenzyl) diethylenetriamine N1,N2,N3,N3-tetraacetic acid (DTTA). The DTTA group forms a stable complex with Eu3+, and the isothiocynate group reacts with amino groups on the protein at alkaline pH to form a stable, covalent thio-urea bond. HERCEPTIN® (0.2 mg in 200 mL sodium bicarbonate buffer pH 9.3) was labeled with 0.2 mg of labeling agent at 4° C. overnight. Eu-labeled HERCEPTIN® was purified by spin column using 50 mmol/L tris-HCl pH 7.5 and 0.9% NaCl elution buffer.

The Eu-HERCEPTIN® binding assay was performed by incubating 0.5-1 million BT-474 or SK-BR3 breast cancer cells per well in a 96-well plate with 2-5 nM Eu-HERCEPTIN® in the presence of various concentrations of unlabeled HERCEPTIN®-based antibodies containing MRDs or HERCEPTIN® for 1 hour at RT. Unbound Eu-HERCEPTIN® was removed by washing using 200 μL complete medium. Cells were then resuspended in 100 μL complete medium and 80 μL of cell suspension transferred to a 96-well isoplate. Cells were incubated with 100 μL Delfia enhancer solution at RT for 10 minutes and cell bound Eu-HERCEPTIN® was detected by Envison (Perkin Elmer).

The inhibition of binding curves obtained using BT-474 cells are presented in FIG. 23. Eu-HERCEPTIN® binding to BT-474 was inhibited by HERCEPTIN® and HERCEPTIN®-based antibodies containing MRDs in a dose-dependent fashion. Comparable IC-50 values were observed: 4.7 nM for HER-lm32(H), 5.7 nM for HER-lm32(L), and 3.7 nM for unlabeled HERCEPTIN®.

Example 28. Inhibition of Breast Cancer Cells Proliferation by HERCEPTIN®-Based Antibodies Containing MRDs

HERCEPTIN sensitive breast cancer cells SK-BR-3 expressing HER2neo receptor were also tested in a bioassay. SK-BR-3 cells (2000 cell/well) were plated in 96 well plates (Costar) in complete McCoy's growth medium containing 2 mM glutamine, pen/strep (Invitrogen) and 10% FBS (HyClone). The cells were cultured for 24 hours at 37° C., 5% CO₂, 85% humidity. On the following day, the growth medium was replaced with starvation medium (McCoy's medium containing 2 mM glutamine, pen/strep, 0.5% FBS). Nine serial dilutions (concentration range 5000-7.8 ng/ml) of HERCEPTIN® and HERCEPTIN®-based antibodies containing MRDs were prepared in complete growth medium. After 24 hours of incubation, the starvation medium was removed, and the serial dilutions of HERCEPTIN® and HERCEPTIN®-based antibodies containing MRDs were transferred to the plates in triplicates. The cells were cultured for 6 days. The proliferation was quantified using the CellTiter Glo luminescence method.

The IC50 values determined using a four-parameter logistic model were as follows: 0.49+/−0.17 nm for HER-lm32(H), 0.81+/−0.19 nm for HER-lm32(L), and 0.67+/−0.15 nm for HER-con4(H). All tested HERCEPTIN®-based antibodies containing MRDs were able to inhibit the proliferation of the SK-BR-3 breast carcinoma cells with subnanomolar IC-50 values. The representative fitted dose response curves shown in FIGS. 24A-C demonstrate that HERCEPTIN®-based antibodies containing MRDs inhibit cell proliferation with similar potency to HERCEPTIN®.

Example 29. Antibody Dependent Cytotoxicity of HERCEPTIN®-Based Antibodies Containing MRDs

To assess the ability of antibodies containing MRDs to mediate ADCC in vitro, a cytotoxicity assay based on the “DELFIA EUTDA Cytotoxicity reagents AD0116” kit (PerkinElmer) was used. In this assay, the target cells were labeled with a hydrophobic fluorescence enhancing ligand (BADTA, bis (acetoxymethyl) 2,2′:6′,2″-terpyridine-6,6″-dicarboxylate). Upon entering the cells, BADTA is converted to a hydrophilic compound (TDA, 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid) by cytoplasmic esterases mediated cleavage and no longer can cross the membrane. After cell lysis, TDA is released into a medium containing Eu3+ solution to form a fluorescent chelate (EuTDA). The fluorescence intensity is directly proportional to the number of lysed cells.

HERCEPTIN® and HERCEPTIN®-based antibodies containing MRDs can mediate ADCC on Her2 positive breast cancer cells by binding to the HER2 receptor on the surface of the target cells and activating the effector cells present in human PBMCs by interacting with their FcγRIII receptors. A HER2 positive human breast cancer cell line SK-BR-3 was used as a target cell line in the ADCC assay to demonstrate this.

SK-BR-3 cells were detached with 0.05% trypsin-versene and resuspended at 1×10⁶ cells/mL in RPMI1640 medium containing 2 mM glutamine, pen/strep and 10% FBS (complete growth medium). 2×10⁶ cells in 2 mL of media were transferred into 15 mL tube and 10 μl of BADTA reagent was added. The cell suspension was mixed gently and placed in the incubator at 37° C., 5% CO₂ and 85% humidity for 15 minutes. Seven 10× serial dilutions starting with 5 μg/mL of HERCEPTIN® or HERCEPTIN®-based antibodies containing MRDs were prepared during cell labeling.

After incubation with BADTA, cells were washed 4 times in complete growth medium containing 2.5 mM Probenecid. Between washes, cells were spun down by centrifugation at 1000 rpm for 3 minutes. After the last wash, labeled SK-BR-3 cells were resuspended in 10 mL complete growth medium and 50 μl of cells were added to each well of 96 well plate, except background wells. 50 μl of serial dilutions of HERCEPTIN® or HERCEPTIN®-based antibodies containing MRDs were added to the designated wells. The plates were transferred to the incubator at 37° C., 5% CO₂ and 85% humidity for 30 minutes.

PBMCs that were purified from human peripheral blood one day prior the ADCC assay, were washed once in RPMI1640 with 2 mM glutamine, pen/strep, 10% FBS. 10 mL of the PBMCs suspension with 2.5×10⁶ cells/mL was prepared. 100 μl of PBMC suspension was transferred into wells containing target cells and HERCEPTIN® or HERCEPTIN®-based antibodies containing MRDs in triplicate. The following controls were placed in designated wells: Spontaneous release (target cells without effector cells), Maximum release (lysed target cells) and Background (media without cells). The plates were incubated for 2.5 hours an incubator with 37° C., 5% CO₂ and 85% humidity.

After incubation 20 μl of the supernatant was transferred to another plate and 200 μl of Europium solution was added. The plates were incubated on a plate shaker at RT for 15 minutes. The time resolved fluorescence was measured using PerkinElmer EnVision 2104 Multilabel Reader.

The following formula was used to calculate percentage of Specific release: Experimental release (counts)−Spontaneous release (counts)×100 Maximum release (counts)−Spontaneous release (counts)

The IC50 values calculated by a four-parameter logistic model were as follows: 0.213+/−0.077 nM for HER-lm32(H), 0.204+/−0.036 nM for HER-lm32(L), and 0.067+/−0.015 nM for HER-con4(H). All tested antibodies containing MRDs demonstrated robust ADCC activity with subnanomolar IC-50 values. The representative fitted dose response curves shown in FIGS. 25A and 25B demonstrate that antibodies containing MRDs are able to mediate cell dependent cytotoxicity with comparable potency to HERCEPTIN®.

A similar experiment was conducted in the presence of Ang2. Human PBMCs were activated with 20 ng/ml of IL2 overnight and added to freshly plated (10,000 cells/well) BADTA labeled SK-BR-3 cells. The effector/target ratio was 25/1. After a 4-hour incubation with serial dilutions of HER-lm32(H) or HUMIRA in the presence of 2 μg/ml Ang2, Eu was added to the medium and TRFI measured on Envision reader (Perkin-Elmer). HER-lm32 was more potent in mediating ADCC in the presence of Ang2.

Example 30. Inhibition of Endothelial Cell Proliferation by AVA-lm32(H)

The biological activities of the AVASTIN®-based antibodies containing MRDs AVA-lm32(H) were tested to determine if they could inhibit VEGF-induced proliferation of Human Umbilical Vein Endothelial Cells (HUVEC) assay.

HUVEC were obtained from GlycoTech (Gaithersburg, Md.) and Lonza on passage 1 and passage 3 respectively. Cells were grown on Endothelial cell basal medium (EBM-2) with addition of 2% fetal bovine serum (FBS) and single quotes (Lonza) at 37° C., 5% CO₂, 85% humidity. For inhibition of proliferation experiments, cells were plated in 96-well plates (Costar) at 2000 cells per well in EBM-2 medium with 2% FBS and cultivated for 24 hours. Nine serial dilutions of AVASTIN® or AVA-lm32(H) were prepared starting with 5 μg/mL on EBM-2 medium with 2% FBS. VEGF (R & D Systems) was added at a final concentration of 10 ng/mL to all serial dilutions. After incubation for 15 minutes at 37° C., 5% CO₂, 85% humidity, serial dilutions were added to the cells. After 96 hours, CellTiter Glo was added to the cells. After incubation at RT for 15 minutes, the cell suspension was transferred into 96 well white opaque plates, and luminescence was measured using PerkinElmer EnVision 2104 Multilabel Reader.

As shown in FIGS. 26A and 26B, AVA-lm32(H) exhibited dose dependent anti-proliferative activity on HUVECs from both sources. IC50 values calculated from 4 PL fitted curves indicate similar potency for AVA-lm32(H) and AVASTIN® (IC50 values 0.36+/−0.42 nM and 0.33+/−0.38 nM, respectively).

Example 31. MRD-Containing Antibodies Inhibit Tumor Proliferation In Vivo

In order to determine the effectiveness of multivalent and multispecific compositions (e.g., MRD-containing antibodies) in vivo, their efficacy in a mouse Colo5 tumor model was assessed. In these experiments, tumors were implanted into the right flank of six-week old female athymic nude mice by injecting 5×10⁶ Colo205 cells suspended in 100 μL PBS. Three groups of eight animals each received intraperitoneal injections of 5 mg/kg of antibody (HERCEPTIN, Rituxan) or an MRD-containing antibody (HER-2×Con4; “H2×Con4”) in 100 μL PBS every third day starting at day 6 after tumor implantation. The results, shown in FIG. 27, demonstrate that the MRD-containing antibody was more efficient at inhibiting tumor growth than either Rituximab® or HERCEPTIN®.

HERCEPTIN with lm32 fused to the C-terminus of the heavy chain also inhibited tumor growth in both Her2 dependent and angiogenesis dependent xenograft tumor models. The HERCEPTIN-lm32 fusion had a similar PK to HERCEPTIN in both mice and monkeys after single dose injections. Furthermore, the HERCEPTIN-lm32 fusion was stable in whole blood at 37° C. for up to 72 hours.

Example 32. Molecular Assays to Evaluate MRD-Containing Antibodies

Novel multivalent and multispecific compositions (e.g., MRD-containing antibodies) are generated by altering the sequence of the MRD and/or the antibody, by altering the location at which the antibody is linked to the MRD, and/or by altering the linker through which the MRD is connected to the antibody. The binding potential, structure, and functional properties of the multivalent and multispecific compositions (e.g., MRD-containing antibodies) are evaluated using known techniques to measure protein binding and function. The multivalent and multispecific compositions (e.g., MRD-containing antibodies) are compared to the MRD alone, the antibody alone, and to other multivalent and multispecific compositions (e.g., MRD-containing antibodies).

An MRD-containing antibody is tested using a solid phase assay in which a target of the MRD and/or antibody is immobilized on a solid surface and then exposed to increasing concentrations of a flourescently labeled MRD-containing antibody. The solid surface is washed to remove unbound MRD-containing antibody and the amount of target-bound MRD-containing antibody is determined directly by quantitating fluorescence. In another experiment, the immobilized target is exposed to increasing concentrations of an unlabeled MRD-containing antibody and the amount of target-bound MRD-containing antibody is determined indirectly by use of a labeled reagent that binds to the MRD-containing antibody

An MRD-containing antibody is tested using a liquid phase assay in which a target of the MRD and/or antibody is added to various concentrations of an MRD-containing antibody is a solution. The interaction of the target with the MRD-containing antibody is detected by the appearance of a molecular complex comprised of a target and MRD-containing antibody that differs in molecular mass (and mobility) from unbound target and unbound MRD-containing antibody.

An MRD-containing antibody is also assayed in a cell based assay in which target-expressing cells are incubated in the presence of increasing concentrations of MRD-containing antibody. The binding of the MRD-containing antibody is detected by fluorescence activated cell sorting. In addition, cellular proliferation, cellular differentiation, protein phosphorylation, protein expression, mRNA expression, membrane composition, signaling pathway activity, and cellular viability are assessed.

Useful multivalent and multispecific compositions (e.g., MRD-containing antibodies) bind to both the MRD target and to the antibody target. In addition, useful multivalent and multispecific compositions (e.g., MRD-containing antibodies) affect at least one cellular process.

Example 33. Identification of MRDs with Improved Characteristics

Two potential T cell epitopes were identified in LM32. In order to identify LM32 variants that did not contain T cell epitopes, and therefore, were less likely to produce immunogenic responses, mutational and deletional variants of the LM32 peptide were created. The LM32 variants listed in Table 7 MRDs were expressed as MBP fusion proteins and tested for the ability to bind Ang2.

TABLE 7 SEQ EC50 ID MRD expressed as a MBP fusion protein (nM) NO KSLSLSPGSGGGSMGAQTNFMPMDNDELLLYEQFI 1.080 32 KSLSLSPGSGGGSMGAQTNFMPMDNEELLLYEQFI 20.700 33 KSLSLSPGSGGGSMGAQTNFMPMDNDEGLLYEQFILQQGLE 1.040 88 KSLSLSPGSGGGSMGAQTNFMPMDNDELGLYEQFILQQGLE na 89 KSLSLSPGSGGGSMGAQTNFMPMDNDEALLYEQFILQQGLE 0.182 90 KSLSLSPGSGGGSMGAQTNFMPMDNDELTLYEQFILQQGLE 1.420 91 KSLSLSPGSGGGSMGAQTNFMPMDNDELLLYEQFIYQQGLE na 92 KSLSLSPGSGGGSMGAQTNFMPMDNDEGLLYEQFIYQQGLE 0.902 93 KSLSLSPGSGGGSMGAQTNFMPMDNDEALLYEQFIYQQGLE 0.392 94 KSLSLSPGSGGGSMGAQTNFMPMDNEELTLYEQFIFQQG na 95 KSLSLSPGSGGGSMGAQTNFMPMDNDEGLLYEEFILQQGLE 0.922 96 KSLSLSPGSGGGSMGAQTNFMPMDNDEALLYEEFILQQGLE 0.426 97 KSLSLSPGSGGGSMGAQTNFMPMDNEELTLYEEFILQQGLE na 98 KSLSLSPGSGGGSMGAQTNFMPMDQDELLLYEQFILQQGLE 0.383 99 KSLSLSPGSGGGSMGAQTNFMPMDDDELLLYEQFILQQGLE 0.240 100

The LM32 variants are then tested for their ability to induce proliferation and/or cytokine release. LM32 variants that are functionally active and have reduced immunogenic potential are identified. An MRD-containing antibody comprising the LM32 variant fused to the light chain of HERCEPTIN®, an MRD-containing antibody comprising the LM32 variant fused to the heavy chain of HERCEPTIN®, an MRD-containing antibody comprising the LM32 variant fused to the light chain of HUMIRA®, an MRD-containing antibody comprising the LM32 variant fused to the heavy chain of HUMIRA®, MRD-containing antibody comprising the LM32 variant fused to the light chain of AVASTIN®, and an MRD-containing antibody comprising the LM32 variant fused to the heavy chain of AVASTIN® are created. The LM32-variant containing antibodies are administered to animal models and the plasma protein representation and plasma and tissue residence are measured and compared to those of HERCEPTIN®, HUMIRA®, and AVASTIN®. In addition, the effects of the LM32-variant containing antibodies on cellular proliferation, angiogenesis, tumorigenicity, arthritic indicators are compared to the effects of HERCEPTIN®, HUMIRA®, and AVASTIN®.

Example 34. In Vivo Assays to Evaluate MRD-Containing Antibodies

In order to determine the efficacy of multivalent and multispecific compositions (e.g., MRD-containing antibodies) in vivo, animal models are treated with an antibody and an MRD-containing antibody and the results are compared.

MRD-containing anti-HER2 antibodies are tested in the following in vivo model. NIH 3T3 cells transfected with a HER2 expression plasmid are injected into nu/nu athymic mice subcutaneously at a dose of 10⁶ cells in 0.1 ml of phosphate-buffered saline as described in U.S. Pat. No. 6,399,063, which is herein incorporated by reference in its entirety. On days, 0, 1, 5, and every 4 days thereafter 100 μg of a HER2 antibody, an ang2-containing HER2 antibody, an igf1r-containing HER2 antibody and an ang2-igf1r-containing HER2 antibody are injected intraperitoneally. Tumor occurrence and size are monitored for one month. Increases in efficacy of multivalent and multispecific compositions (e.g., MRD-containing antibodies) compared to antibodies are observed.

MRD-containing anti-VEGF antibodies are tested in the following in vivo model. RIP-TβAg mice are provided with high-sugar chow and 5% sugar water as described in U.S. Published Application No. 2008/0248033, which is herein incorporated by reference in its entirety. At 9-9.5 or 11-12 weeks of age, the mice are treated twice-weekly with intra-peritoneal injections of 5 mg/kg of an anti-VEGF antibody, ang2-containing VEGF antibody, ifg1r-containing VEGF antibody or ang2- and igf1r-containing antibody. The 9-9.5 week mice are treating for 14 days and then examined. The 11-12 week mice are examined after 7, 14, and 21 days of treatment. The pancreas and spleen of the mice are removed and analyzed. Tumor number is determined by dissecting out each spherical tumor and counting. Tumor burden is determined by calculating the sum of the volume of all tumors within the pancreas of a mouse. The effect on angiogenesis is determined by calculating the mean number of angiogenic islets observed. Increases in efficacy of multivalent and multispecific compositions (e.g., MRD-containing antibodies) compared to antibodies are observed.

MRD-containing anti-TNF antibodies are tested in the following in vivo model. Transgenic mice (Tg197) are treated with three intra-peritoneal injections of anti-TNF antibody or ang2-containing TNF antibody at 1.5 μg/g, 15 μg/g, or 30 μg/g as in U.S. Pat. No. 6,258,562, which is incorporated herein by reference in its entirety. Injections continue for about 10 weeks and macroscopic changes in joint morphology are recorded each week. At 10 weeks, mice are sacrificed and microscopic examination of tissue is performed. Joint size is established as an average measurement on the hind right ankle using a micrometer device and arthritic scores are recorded as follows: 1=no arthritis; +/−=mild (joint distortion); ++=moderate arthritis (swelling, joint deformation); and +++=heavy arthritis (ankylosis detected on flexion and severely impaired movement). Histopathological scoring based on haematoxylinleosin staining of joint sections is based as follows; 0=No detectable disease; 1=proliferation of the synovial membrane; 2=heavy synovial thickening 3=cartilage destruction and bone erosion. Increases in efficacy of multivalent and multispecific compositions (e.g., MRD-containing antibodies) compared to antibodies are observed.

Example 35. MRD-Containing Antibodies are Superior to Combinations of Antibodies and MRDs

In order to compare the efficacy of multivalent and multispecific compositions (e.g., MRD-containing antibodies) to combinations of antibodies and MRDs, their effect on SK-BR-3 cells treated with EGF was studied.

SK-BR-3 cells were treated with HERCEPTIN, HERCEPTIN containing an EGFR-MRD, HUMIRA containing an EGFR-MRD (“MRD alone”), or HERCEPTIN in combination with HUMIRA containing an EGFR-MRD (“antibody plus MRD alone”) for 10 minutes or 3 hours and stimulated with EGF for 5 minutes. Cell lysates were collected and western blots were used to determine phosphotyrosine or phospho-Akt levels. The results are shown in FIG. 28. The HERCEPTIN containing an EGFR-MRD completely inhibited EGF-induced receptor phosphorylation and Akt activation, whereas HERCEPTIN, the MRD alone, and the HERCEPTIN antibody plus MRD alone had little or no effect.

In addition, the effect of multivalent and multispecific compositions (e.g., MRD-containing antibodies) on cellular proliferation was compared to the effect of the combination of antibodies and MRDs. In these experiments, MCF-7 derived breast carcinoma cells were treated with a HERCEPTIN antibody containing an igf1r-targeting MRD fused to the C-terminus of the light chain, HERCEPTIN alone, or a HUMIRA antibody containing the same igf1r-targeting MRD (“MRD alone”). As shown in FIG. 29A, the MRD-containing antibody inhibited cell proliferation better than HERCEPTIN alone or the MRD alone.

MCF-7 cells were also treated with a penta-specific MRD-containing antibody. First, these penta-specific multivalent and multispecific compositions (e.g., MRD-containing antibodies) were shown to bind to five targets. An Ang2-targeting MRD (lm32), and an EGFR-targeting MRD were fused to the N- and C-termini, respectively of a HERCEPTIN antibody. In addition, a αvβ3-targeting MRD (eeti) and an igf1r-targeting MRD were fused to the N- and C-termini, respectively of the same HERCEPTIN antibody. Ang2, ErbB2, EGFR, IGF1R and αvβ3 were coated on separate wells of a 96-well plate and incubated with serial dilutions of the MRD-containing antibody. Bound MRD-containing antibody was detected using anti-human IgG-kappa detector. The results demonstrated that the MRD-containing antibody bound to Ang2, ErbB2, EGFR, IGF1R and αvβ3 with low nanomolar or sub-nanomolar affinities that are comparable to the binding-affinities of the MRDs or antibodies individually.

Then, the ability of these penta-specific multivalent and multispecific compositions (e.g., MRD-containing antibodies) to inhibit proliferation of MCF-7 cells was tested as described above. The results, shown in FIG. 29B, demonstrate that the HERCEPTIN penta-specific multivalent and multispecific compositions (e.g., MRD-containing antibodies) decrease proliferation more efficiently than the HERCEPTIN antibody.

Example 36. MRD-Containing Anti-TNF Antibodies

An MRD-containing antibody that targets TNF was created by fusing an Ang2-binding MRD (lm32) to the C-terminus of HUMIRA heavy chain and was expressed in both transient and stable expression systems. The MRD-containing antibody bound simultaneously to TNFα and Ang2. The MRD-containing antibody bound soluble and cell surface TNFα and retained the binding specificity and Fc functions of HUMIRA (e.g., FcRn, FcgammaR1 and FcgammaR3 binding) and also inhibited Ang2 mediated signaling through Tie-2 receptor in a dose dependent manner with sub-nanomolar affinities.

The HUMIRA-lm32 fusion also inhibited TNFα mediated cytotoxicity in L929 cells. The cells were cultured in 96-well plates overnight and treated with 1 ng/mL TNF plus 1 μg/ml of Actinomycin D in the presence of HUMIRA or HUMIRA-lm32 for 24 hours at 37° C. After incubation, 100 μL of cell Titer-Glow reagent was added, and luminescence was measured using InVision (Perkin-Elmer) after 15 minutes at room temperature. The results demonstrated that HUMIRA and HUMIRA-lm32 displayed equal potency in inhibiting TNFα-mediated cytotoxicity in L929 cells (HUMIRA IC50=19.0 ng/ml; HUMIRA-lm32 IC50=18.9 ng/ml).

Furthermore, the HUMIRA-lm32 fusion displayed a dose-dependent protection of hTNF-transgenic mice from clinical signs of arthritis in a well-established mouse model. See, e.g., Keffer et al., EMBO J. 10:4025-4031 (1991). A single-dose PK study in mice demonstrated that the HUMIRA-lm32 fusion and HUMIRA have similar PK and immunogenicity profiles. However, the HUMIRA-lm32 fusion showed increased efficacy in this model compared to that of HUMIRA alone both when measured by clinical symptoms or by histology. See FIG. 30.

Example 37. Zybodies Inhibit EGF-Induced Signaling

SK-BR3 cells were plated at 0.5×10⁶ cells/well in 6-well plates and incubated (37° C., 5% CO₂) for 24 hours at which time cells were treated with 10 μg/mL bi- and tri-specific zybodies or Herceptin in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37° C. Cells were then stimulated with 100 ng/mL EGF for 5 minutes. After stimulation, cells were was disrupted in 200 μL cell lysis buffer (10 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10% Glycerol, 1 mM sodium vanadate, 5 mM EDTA and protease inhibitors). The cell lysates were centrifuged at 14000 RPM for 10 minutes at 4° C. to remove cell debris. Equal volume of 2× sample buffer and cell lysates were mixed and boiled at 100° C. for 5 minutes, proteins were resolved on a 10% SDS-PAGE and transferred to PVDF (catalog#LC2005, Invitrogen) membranes. Membranes were block with 3% BSA, 0.1% Tween 20 overnight and incubated with antibodies to phospho-AKT (catalog#AF887, R&D systems), phospho-ERK (Catalog#AF1018, R&D systems), and total ERK (MAB1576, R&D system). Horseradish or AP conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) were used to visualize immune-reactive proteins using chemiluminescence or AP detection reagents respectively.

One bispecific antibody used in this example comprised Herceptin and an EGFR-binding MRD (Her-egfr). Another bispecific antibody comprised Herceptin with a Pertuzumab-scfv (which targets a different HER2 epitope than the Herceptin antibody) on the C-terminus of the heavy chain (Her-Pertuzumab(H)). One trispecific antibody comprised Herceptin with an EGFR-binding MRD on the C-terminus of the heavy chain and a Pertuzumab-scfv on the C-terminus of the light chain. Another trispecific antibody comprised Herceptin with an EGFR-binding MRD on the C-terminus of the light chain and a Pertuzumab-scfv on the C-terminus of the heavy chain (Her-zEGFR(L)-Pert(H)). Another trispecific antibody comprised an EGFR-binding MRD, and a Pertuzumab-scfv which targets a different HER2 epitope than the Herceptin antibody (Her-Pert(L)zEGFR(H) and Her-zEGFR(L)-Pert(H).

SK-BR3 cells express very high levels of HER2 and are sensitive to anti-proliferative effects of Herceptin. Inhibition of constitutively activated AKT is one of the mechanisms for the anti-proliferative effects of Herceptin in HER2 over-expressing cells. Such inhibition can be overcome by the addition of growth factors such as EGF, IGF-1 and Heregulin (HRG) through the induction of intracellular pro-mitogenic signaling. As shown in FIG. 31, EGF-induced activation of AKT and ERK pathways in SK-BR3 cells as shown by increase in phoshorylated AKT and ERK levels. Herceptin had no effect on EGF-induced activation of signaling pathways in SK-BR3 cells whereas Her-egfr and the two tri-specific zybodies with an EGFR targeting peptide inhibited EGF effects. Compared to the bi-specific antibody which inhibited only EGF-induced signaling, tri-specific antibodies also inhibited constitutively active levels of Akt and ERK in SK-BR3 cells.

Example 38. Zybodies Inhibit Heregulin-Induced Signaling

SK-BR3 cells were plated at 0.5×10⁶ cells/well in 6-well plates and cultured overnight. The next day, cells were treated with 10 μg/mL bi- and tri-specific zybodies or Herceptin in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37° C. Cells were then stimulated with 200 ng/mL Heregulin for 10 minutes. Western blot analysis was performed to detect phosphorylated and total Aid and ERK levels as described in Example 37.

Heregulin binds to HER3 and induces activation of signaling pathways via HER2-HER3 heterodimer formation. As shown in FIG. 32, Heregulin induced activation of Akt and ERK in SK-BR3 cells. Herceptin and HER-egfr had no effect on Heregulin-induced Akt activation. Pertuzumab, but not Herceptin, blocks Heregulin mediated HER2-HER3 heterodimer formation and signaling. FIG. 32 shows that bi- and tri-specific zybodies comprising a Pertuzumab-scfv completely inhibited Heregulin-induced Akt activation in SK-BR3 cells.

Example 39. Zybodies Inhibit EGF- and Heregulin-Induced Signaling

SK-BR3 cells were plated at 0.5×10⁶ cells/well in 6-well plates and cultured overnight. The next day, cells were treated with 10 μg/mL bi and tri-specific zybodies or Herceptin in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37° C. Cells were then stimulated with 100 ng/mL EGF and 200 ng/mL Heregulin for 10 minutes. Western blot analysis was performed to detect phosphorylated and total Akt and ERK levels as described in Example 37.

Combined stimulation of SK-BR3 cells with EGF and Heregulin resulted in both AKT and ERK activation (FIG. 33). Herceptin, HER-efgr and HER-pertuzuScfv were ineffective in blocking the combined effects of EGF and Heregulin. Tri-specific zybodies that contained both an EGFR targeting peptide and a Pertuzumab-scfv completely inhibited EGF and Heregulin induced Akt and ERK activation in SK-BR3 cells.

Example 40. Zybodies Down-Regulate Cell-Surface EGFR

SK-BR3 cells were plated at 0.5×10⁶ cells/well in 6-well plates and cultured overnight. The next day, cells were treated with 10 μg/mL bi- and tri-specific zybodies, Herceptin, Erbitux, EGF, and combinations thereof in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37° C. as indicated. Cells were detached and the levels of EGFR on the cells were determined by Flow cytometry using PE conjugated anti-EGFR antibody.

The zybodies used in this experiment were a Herceptin antibody with an EGFR-binding MRD on the C-terminal of the heavy chain (HER-egfr(H)); a Humira antibody with an EGFR-binding MRD on the C-terminal of the heavy chain (HUM-egfr(H)); Herceptin with a Pertuzumab-scfv on the C-terminal of the heavy chain (HER-perscfv(H)); Humira with a Pertuzumab-scfv on the C-terminal of the heavy chain (HUM-perscfv(H)); Herceptin with a Pertuzumab-scfv on the C-terminal of the light chain and an EGFR-binding MRD on the C-terminal of the heavy chain (HER-perscfv(L)-egfr(H)); Herceptin with a Pertuzumab-scfv on the C-terminal of the heavy chain and an EGFR-binding MRD on the C-terminal of the light chain (HER-perscfv(H)-egfr(L)); and a Humira antibody with an EGFR-binding MRD (HUM-egfr).

Erbitux, EGF, and bi- and tri-specific zybodies containing egfr-targeting peptides down regulated EGFR receptor levels on the cell surface. FIGS. 34A and B. Treatment with HER-egfr for 24 hr. was more effective (61%) than HUM-egfr alone (27%) or in combination of HUM-egfr plus Herceptin (41%) and Erbitux plus Herceptin (51%) (FIGS. 34A and B). This indicates that simultaneous targeting of EGFR and HER2 in zybody format was more effective in down regulating EGFR than a combination of two individual antibodies. Also, treatment with tri-specific antibodies that target EGFR and two different epitopes of HER2 completely down regulated (99%) EGFR levels on SK-BR3 cells.

Example 41. Zybodies Down-Regulate EGFR Expression

SK-BR3 cells were plated at 0.5×10⁶ cells/well in 6-well plates and cultured overnight. The next day, cells were treated with 10 μg/mL bi and tri-specific zybodies or Herceptin in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37° C. Cell lysates were prepared and Western blot analysis was performed to detect EGFR using EGFR antibody (catalog# MAB10951, R&D system) and total ERK levels as described in Example 37.

Treatment with Herceptin and Her-Pertuzumab scfv had no effect on EGFR levels in SK-BR3 cells. HER-egfr partially down regulated EGFR protein and almost complete degradation of EGFR was observed in cells treated with tri-specific zybodies.

Example 42. Zybodies Increase MRD Potency

The following experiments were performed to examine the effect that antibody fusion has on MRD potency. Individual wells of a 96-well plate were coated with IGF1R and ErbB2-Fc (HER2) (1 ug/ml) at varying ratios. Wells were washed, and then serial dilutions of zybodies were added in the presence of biotin-labeled IGF-1. The zybodies used were a Herceptin antibody containing an IGFR-targeting MRD on the C-terminus of the heavy chain (HER-igfr(H)) and a Humira antibody containing an IGFR-targeting MRD on the C-terminus of the heavy chain (HUM-igfr(H)). Bound IGF-1 was quantitated by addition of streptavidin-HRP. The results are shown in the table below.

TABLE 8 Inhibition of IGF-1 Binding, IC₅₀ (nM) Ratio of IGF1R:HER2 Zybody 1:0 3:1 1:1 1:3 HER-igfr(H) 220 20.91 1.44 0.14 HUM-igfr(H) 240 189.9 186 111

The inhibition of IGF-1 binding was similar for both zybodies tested when the plates did not contain ErbB2-Fc (HER2) (IC50 HER-igfr(H)=220 nM; IC50 HUM-igfr(H)=240 nM). When the plates contained both the antibody target and the MRD target, binding to MRD target drastically decreased. For example, using the HER-igfr(H) zybody, inhibition of IGF-1 binding dropped from IC₅₀=220 nm when the antibody target was not on the plates (ratio of IGFR1R:HER2=1:0) to IC₅₀=1.44 when the antibody target and MRD target were present on the plates in equivalent amounts (ratio of IGFR1R:HER2=1:1). The data indicate that engagement of both antibody and MRD targets by HER-igf1r(H) enhances the potency of the low affinity IGF-1R MRD>1000-fold. In contrast, this drastic effect was not observed when the same experiment was performed using a zybody containing the Humira antibody, which does not bind to HER2 even though the zybody contained the same IGF1R-targeting MRD. Thus, bi-specific zybodies display enhanced MRD potency through heterotypic avidity-driven binding.

Example 43. Conformational Constraints can Increase MRD Binding and Stability

In order to determine the effect of conformational constraints on MRDs in the context of an MRD-containing antibody, several modified MRD constructs containing cysteines at various locations were developed. The cysteines form intermolecular disulfide bonds and therefore contain the three-dimensional conformation of the proteins. The MRD that was altered in this experiment was MPM. The sequence of MPM, which is shown in the table below, is similar to the sequence of lm32 (described above), but contains four amino acid changes: MSG, N16Q, L19A, and Q24E. The G at position five in MPM minimizes potential oxidation of the methionine residue, and L19A and Q24E remove potentially immunogenic sequences. In these experiments, all MRDs were fused to the C-terminus of the Herceptin heavy chain.

The resulting multivalent and multispecific compositions (e.g., MRD-containing antibodies) were then tested for target binding and stability. Stability was determined by administering the MRD-containing antibody by IV injection to mice and comparing the levels of MRD in the plasma 15 minutes post-injection to the levels of MRD in the plasma 2, 3, or 4 days post-injection. The results are shown below in Table 9.

TABLE 9 Binding and Stability of Constrained MRDs EC50 MRD % Construct Sequence nm 48 hrs HER-2x-con4 AQQEECEWDPWTCEHMGSGSATGGS 0.018 70 GSTASSGSGSATHQEECEWDPWTCEH MLE (SEQ ID NO: 136) HER-lm32(H) KSLSLSPGKGGGSMGAQTNFMPMDND 0.033 23 ELLLYEQFILQQGLE (SEQ ID NO: 34) HER-mpm(H) KSLSLSPGSGGGSGGAQTNFMPMDQD 19 EALLYEEFILQQGLE (SEQ ID NO: 56) HER-lm32 (MPM KSLSLSPGSGGGSGGACTNFMPMDQD 4.06 Q8C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 57) HER-lm32 (MPM KSLSLSPGSGGGSGGAQCNFMPMDQD 0.598 38 T9C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 58) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTCFMPMDQD 0.177 9 N10C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 67) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTNCMPMDQD 5.91 F11C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 68) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTCFMPMDQD 0.403 33 N10C L28C) (H) EALLYEEFICQQGLE (SEQ ID NO: 69) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTNFMPMDQD 0.0641 60 M5C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 70) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTNFMPMDQD 0.109 14 M5C L28C) (H) EALLYEEFICQQGLE (SEQ ID NO: 101) HER-lm32 MPM KSLSLSPGSGGGSGGCQTNFMPMDQD 0.174 82 A7C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 108) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTNFMCMDQD P13C G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 109) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTCFMPMDQD 0.2 13 M5C N10C) (H) EALLYEEFILQQGLE (SEQ ID NO: 110) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTNCMPMDQD M5C F11C) (H) EALLYEEFILQQGLE (SEQ ID NO: 111) HER-lm32 (MPM KSLSLSPGSGGGSCFMPMDQDEALLYE 0.751 3 D5 N10C G30C) (H) EFILQQCLE (SEQ ID NO: 112) HER-lm32 (MPM KSLSLSPGSGGGSCFMPMDQDEALLYE 0.697 36 D5 N10C L28C) (H) EFICQQGLE (SEQ ID NO: 113)

The results demonstrated that adding two cysteine residues outside the core target-binding domain (e.g., PMDQDEALLY in MPM) of an MRD can increase the MRD half-life without substantially decreasing the binding affinity.

Constructs containing one cysteine located near the terminus of the molecule (e.g., about two amino acids away from the terminus) and one cysteine located on the opposite end of the target-binding domain (e.g., at least about 3 or about 4-7 amino acids outside of the core binding domain) and near the protein fusion (e.g., about 4-6 amino acids away from the linker or antibody sequence) can show increased MRD half-life without substantially decreasing the binding affinity. Furthermore, MRDs that include cysteines within the target-binding site, in particular on either end of the target-binding site can be MRDs that have both high stability and efficient target binding.

Novel multivalent and multispecific compositions (e.g., MRD-containing antibodies) in which the MRDs have a long half-life in vivo and efficient target-binding can be identified by altering the sequence of the MRD to include at least two cysteines. The cysteines can be at least about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids away from each other. The binding potential and half-life of the MRD in the MRD-containing antibody is evaluated using known techniques and the methods described herein. MRD-containing antibodies are administered to mice intravenously. Plasma is collected from mice shortly after administration (e.g., 15 minutes after administration) and at a later time point (e.g., 2, 3, or 4 days after administration). Useful multivalent and multispecific compositions (e.g., MRD-containing antibodies) that both bind efficiently to the MRD target and are stable in vivo (e.g., at least about 50% of the MRD is present 48 hours after administration) are identified.

Example 44. MRD-Containing Antibodies for Redirected T-Cell Killing

Antibody MRD-fusion molecules were prepared by fusion of a CD3-targeting peptide to an anti-CD19 antibody and by fusion of a CD19-targeting peptide to an anti-CD3 antibody. The resulting MRD-containing antibodies are analyzed by flow cytometric analysis on CD3-positive Jurkat cells, human PBMCs and a number of different CD19-positive B cell lymphoma cell lines (e.g., SKW6.4, Blin I, BJAB, Daudi and Raji) to determine their specific binding affinities to each target. Since BL60 and the plasmacytoma cell lines NCI and L363 are negative for both CD3 and CD19, they are used as negative control cells to determine the specificity MRD-containing antibodies. CD3-negative Jurkat cells can also be used as a negative control cell population. Cell lines are cultured in complete RPMI 1640 (Invitrogen) with 10% FCS (GIBCO).

Cells are washed with PBS and blocked by resuspension in PBS with 10% human IgG (Innovative Research) and 0.1% NaN₁ (blocking buffer) for 30 min at 4° C. Cells are then pelleted by centrifugation (100×g for 5 min) followed by incubation with the MRD-containing antibodies in blocking buffer for 30 min at 4° C. The cells are washed three times with PBS, and cell-surface bound MRD-containing antibodies are detected. Flow cytometry can be performed with a BD FACScan.

Example 45. In Vitro Cytotoxicity of MRD-Containing Antibodies for Redirected T-Cell Killing

The bispecific CD19/CD3 MRD-containing antibodies are assayed with respect to their abilities to induce redirected T-cell killing of CD19-positive lymphoma cells. Human peripheral blood mononuclear cells (PBMCs) are isolated as effector cells from fresh buffy coats of random donors using Lymphoprep™ (Nycomed/Axis-Shield PoC) gradient centrifugation with subsequent centrifugation at 100×g to remove platelets. CD19-positive B cells are depleted using Dynabeads® CD19 Pan B (Life Technologies). The PBMC populations are analyzed by flow cytometry before and after CD19-positive B cell depletion by labeling with FITC-conjugated mouse antibody against human CD19 and counter-labeled with a PE-conjugated anti-CD45 antibody. The PBMCs are incubated overnight at 37° C. under 5% CO₂. CD19-positive B cell lines (e.g., SKW6.4, Blin I, BJAB, Daudi and Raji) were used as target cells.

Target cells are incubated in 96-well plates using RPMI 1640 complete medium (Invitrogen) with 10% FCS (GIBCO) at different densities, such that addition of the same number of unstimulated PBMCs resulted in different effector-to-target cell (E:T) ratios. Various concentrations of bispecific CD19/CD3 MRD-containing antibodies are then added to each well followed by the addition of unstimulated PBMCs. Plates are incubated at 37° C. under 5% CO₂ for 3 hrs. Cytotoxicity can be measured using the DELFIA® EuTDA cytotoxicity assay (PerkinElmer) in round-bottom 96-well-plates following manufacturer's instructions. Spontaneous cell death is measured by incubating the target cells without effector cells or MRD-containing antibodies, and maximal cell death is determined by incubating the target cells with 10% Triton X-100. The fraction of specific cell lysis is calculated as the ratio between effector mediated cytotoxicity ([experimental cell death]−[spontaneous cell death]) and the maximum expected cytotoxicity ([maximal cell death]−[spontaneous cell death]).

Example 45. In Viva Efficacy of MRD-Containing Antibodies for Redirected T-Cell Killing

Raji B lymphoma cells are removed from routine cell culture, washed in PBS, and prepared as 1×10⁷ cells/ml. NOD/SCID mice are then inoculated subcutaneously with 1×10⁶ Raji cells with or without 5×10⁶ PBMCs (as prepared above) in a 50% Matrigel solution. Bispecific CD19/CD3 MRD-containing antibodies are administered intravenously 1 hr after lymphoma cell inoculation. As negative controls, bispecific MRD-containing antibodies directed to HER2 and CD3 (i.e., a CD3-binding MRD fused to an anti-HER2 antibody and a HER2-binding MRD fused to an anti-CD3 antibody) and HER2 and CD19 (i.e., a CD19-binding MRD fused to an anti-HER2 antibody and a HER2-binding MRD fused to an anti-CD19 antibody) and PBS are also administered intravenously 1 hr after lymphoma cell inoculation. MRD-containing antibodies or PBS are administered once per day for four days after the initial dose. Subcutaneous tumors are measured by caliper to determine growth rate for each treatment group. Body weight of mice is also determined twice per week as an indicator of treatment tolerability.

Example 46. Zybodies Displaying DR5 Agonizing MRDs on Distinct mAb Scaffolds

The cytotoxic effects of several zybodies (i.e., MRD-containing antibodies) expressing different valencies of a DR5 (TNFRSF10B/TRAILR2) agonizing MRD were assessed on three different mAb scaffolds using a cell viability assay. Briefly, eight serial dilutions of Zybodies (5-fold, starting at 30 mg/mL) were incubated with 10⁴ COLO-205 cells in 96-well plate wells, containing 100 ml of RPMI medium supplemented with 10% FBS. After 48 hrs cell viability was measured by Cell TiterGlo® (Promega). The results depicted in FIG. 36A, demonstrate that a tetravalent 2×dr51m(H)-TRA zybody, containing four copies of the DR5 MRD fused to the trastuzumab mAb scaffold, demonstrated higher cytotoxic activity compared to bivalent Zybodies dr51m(H)-TRA, dr51m(L)-TRA and TRA-dr51m(H). These results demonstrate that increased valency of the DR5 agonistic MRDs correlates with increased cell killing through the DR5 death-domain receptor.

To further investigate the role of valency on DR5-mediated cell cytotoxicity, two octa-valent zybodies (2×dr51m(L)2×dr51m(H)-HER and 2×dr51m(L)2×dr51m(H)-PAL comprising a trastuzumab and palivizumab mAb scaffold, respectively) were assayed for their ability to kill COLO205 tumor cells compared to a similarly constructed tetra-valent zybody of the same specificities.

The octavalent zybodies 2×dr51m(L)2×dr51m(H)-HER and 2×dr51m(L)2×dr51 m(H)-PAL, containing eight copies of the DR5 MRD, demonstrated higher cytotoxic activity compared to tetravalent zybodies 2×dr51m(H)-HER and 2×dr51m(H)-PAL (FIGS. 36B-C). These results further establish the correlation between increase DR5-binding valency and increased cytotoxic activity (i.e., cell death).

To explore the effect of additional non-DR5 binding specificities on the cytotoxicity of DR5-based zybodies, the Her2-targeting MRD (eb18) was fused to the C-terminus of the heavy chain of trastuzumab to yield a tri-specific, tetra-valent DR5 zybody, 2×DR5(H)-TRA-eb18(H). The eb18 MRD was chosen because it binds her2 through an epitope that is distinct from that recognized by trastuzumab. The multi-epitopic targeting of her2 enhanced the cytotoxicity of the zybody (FIG. 36D). These results demonstrate that a zybody capable of multi-epitopic targeting of non-DR5 receptors (her2) enhances the cytotoxic activity of a tetra-valent DR5 targeting MRDs.

To further explore the effect of multi-specificity on the cytotoxic activity of DR5-based zybodies, the EGFR-targeting MRD (eb13) was fused to the C-terminus of the heavy chain of cetuximab (anti-EGFR) to yield a tri-specific, tetra-valent DR5 zybody, 2×DR5(H)-CET-eb13(H). The eb13 MRD was chosen because it binds EGFR through an epitope that is distinct from that recognized by cetuximab. The multi-epitopic targeting of EGFR enhanced the cytotoxicity of the zybody (FIG. 36E). These results demonstrate that a zybody capable of multi-epitopic targeting of non-DR5 receptors (EGFR) enhances the cytotoxic activity of a tetra-valent DR5 targeting MRDs.

To investigate the effect of mAB scaffold specificity on the activity of DR5-based zybodies, the cytotoxic potency of three zybodies was compared where each zybody was constructed with the same DR5-targeting MRDs on a different scaffold; cetuximab (CET), trastuzumab (TRA), and palivizumab (PAL). The results demonstrate that the mab scaffold of the zybody can influence the apparent cytotoxic potency of DR5 targeting MRDs (FIG. 36F).

Example 47. Specificity of Ang-Binding MRDs to Ang1, Ang2, and Ang4

The specificity of a select group of Ang2-binding peptides displayed on phage for Ang1, Ang2 and Ang4 was assessed in a direct binding ELISA. Briefly, ELISA plates were coated with the indicated protein at 0.1 ug/ml in PBS. Plates were blocked and phage supernatants added at a dilution of 1:10 in blocking buffer. Unbound phage were removed by washing with PBS 0.1% Tween and bound phage detected using an HRP conjugated anti-M13 monoclonal antibody. The assayed peptides were as follows: ANG100 (SEQ ID NO:621), ANG126 (SEQ ID NO:232); ANG129 (SEQ ID NO:234); ANG156 (SEQ ID NO:255); ANG157 (SEQ ID NO:256); ANG163 (SEQ ID NO:261); ANG179 (SEQ ID NO:270); ANG200 (SEQ ID NO:628); ANG303 (SEQ ID NO:331); ANG318 (SEQ ID NO:625); ANG335 (SEQ ID NO:332); and ANG599 (SEQ ID NO:367).

The data demonstrate that all Ang-binding peptides bind Ang2 but they differ in the relative binding to Ang1 and Ang4 (FIG. 37). These data demonstrate that the fine specificity of Ang binding peptides can differ from one peptide to another. The cross-reactivity of MRDs with members of the Ang family can be exploited in diseases characterized by differential Ang-family member expression.

Example 48. Identification of Exemplary Target Binding MRDs

The following exemplary target-binding MRDs were identified using procedures essentially as descried herein.

TABLE 10 ANG2 binding MRDs SEQ ID: Ref. No. Peptide Sequence 136 ANG2 SACDHPMNNKCG 137 ANG4 SACGTFTHPQCG 138 ANG6 SACHPMAPKSCG 139 ANG7 SACGSYNHPMCG 140 ANG8 SACGSYEHPMCG 141 ANG10 SACQSSVCFFVRWRF 142 ANG12 SACHPLIHNRCG 143 ANG18 TCIFNFPACI 144 ANG19 FCDEYTPAWCW 145 ANG20 MCPDFGMFIYCI 146 ANG22 QCQEVISACFGQAGQGGGSGGS QCWLMELVCL 147 ANG23 KCPDMWPQCW 148 ANG24 KCPDMWPMCW 149 ANG25 QCEWIWVACDGQAGPAGFCYE HMLMCA 150 ANG26 RCYEVWWDCA 151 ANG27 ACIPDLFLCM 152 ANG28 QCMWMWYQCR 153 ANG29 KCPDMWPECW 154 ANG30 PCQYWWVLCP 155 ANG31 LCWNHWWMCE 156 ANG32 FCHEYTPACCW 157 ANG33 KCPDFWPECW 158 ANG34 ECYHHWPECM 159 ANG35 RCPDMWFPACW 160 ANG37 ICIYWKDPWCY 161 ANG38 KCPDLWPQCY 162 ANG40 YCWVIAKSCVGQAGQGGGGGG SICRHYVWRCE 163 ANG41 LCWNHWWMCD 164 ANG42 MCTWQFMVCPGQAGQGGGSG GSYCWEYVYRCD 165 ANG43 MCWNHWWMCE 166 ANG46 HCIFNFPACI 167 ANG47 LCFNHWWMCP 168 ANG49 MCIYWKEPWCY 169 ANG50 WCDHTEAWPECF 170 ANG51 ECYWYPNPMWCY 171 ANG52 HCPWGTTMNCV 172 ANG54 ECTDYPEWRYCQ 173 ANG55 ECFFFPNPWHCY 174 ANG56 PCAWFPNPSFCY 175 ANG58 MCPWWYDHLHCY 176 ANG59 MCPIWYDHQWCH 177 ANG61 ECWWYYNHFWCH 178 ANG62 ECAWFPNPWCCY 179 ANG63 GCWHDGFMCF 180 ANG64 WCDHWIVWPECW 181 ANG65 QCNDYPNWRYCF 182 ANG66 PCAWYPNPEFCY 183 ANG67 LQYEEFYDHV 184 ANG68 QCPYRINHQRCS 185 ANG69 DCVWFPNPDWCY 186 ANG70 FCALFPYMFPCE 187 ANG71 DGSPCAWFPNPSFCY 188 ANG72 VCPWWYDHWLCS 189 ANG73 ECDGWEYHYFCW 190 ANG74 YCPLCQYHYGCF 191 ANG75 SCWWYPNPQWCY 192 ANG76 ECYFLVNMEWCQ 193 ANG77 TCWFWELEFFCI 194 ANG78 KCYWFPNPIECY 195 ANG79 DCAWFPNPAWCH 196 ANG80 HCPYWYDHELCH 197 ANG81 YCPQWYDHWLCT 198 ANG82 NCWEYPNPAWCY 199 ANG83 ECDGWEYHYVCW 200 ANG84 ECYWYPNPWFCY 201 ANG85 DCVDYPGWEYCV 202 ANG86 ECYMLHACYDEGH 203 ANG87 WCDRGTYVMFCD 204 ANG88 TCPVWWDDYWCG 205 ANG89 WCDYGTYVVQCE 206 ANG90 HCYWHPSPSWCY 207 ANG91 QCYWFPNPLECY 208 ANG93 QCKWFPNPKECY 209 ANG94 PCPYYFNRQHCV 210 ANG95 ICYVYGLEEWCI 211 ANG98 HCPLWYDHYFCA 212 ANG101 ACAWYPNPVECY 213 ANG102 ECAIYPNPQWCY 214 ANG103 DCYYYPNPSWCW 215 ANG104 DCYYPPNPQWCF 216 ANG106 QCPWWYDHYWCN 217 ANG107 FCDFYDYHYVCF 218 ANG108 VCPYWWDHWLCL 219 ANG109 MCPFWYDHELCV 220 ANG110 ICPWWYDHYTCF 221 ANG111 ACDWFQNPYVCW 222 ANG112 PCAWYPNPAECY 223 ANG113 ECPFWYDHYMCM 224 ANG114 PCHWFPNPTECY 225 ANG116 ECYTFPNPQFCY 226 ANG118 SCYSYPNPAWCY 227 ANG119 ECYWYPNPHWCR 228 ANG120 IICYWIIPSPNWCR 229 ANG121 HCPDWYNHQLCP 230 ANG122 ACVWFPNPDWCY 231 ANG123 LGPVLLECYFYPNPQHCY 232 ANG126 VPIDCYFYPNPPECYLHN 233 ANG128 GYRDCYFYPNPRHCYGSH 234 ANG129 VVVEFWECYVYPNPPHCY 235 ANG130 LVNDCFFFPNPPHCYVSS 236 ANG131 RMADCYFYPNPQHCYTEG 237 ANG132 DEQACYFYPNPAHCYQRD 238 ANG133 FLGAMMDCYFFPNPPHCY 239 ANG134 VYQDCYNFPNPVIICYRTT 240 ANG135 LVDYSMDCYFFPNPPHCY 241 ANG136 HSLIYHDCYFYPNPPHCY 242 ANG137 LLSVLLECYFYPNPPHCY 243 ANG138 PTDVLLECYLYPNPPHCY 244 ANG139 SLPLRAFCYFPPNWPHCY 245 ANG142 TDTDCYFFPNPPHCYSPG 246 ANG143 IYDDCYIFPNPHHCYVGF 247 ANG144 PWHECYWYPNPPHCYGIL 248 ANG145 MPLIFSECYSYPNPPHCY 249 ANG148 GNFAHEECYFYPNPAHCY 250 ANG149 QPRLLHDCYFYPNPSHCY 251 ANG150 NPTDCYFYPNPQHCYNWG 252 ANG151 YPFFLVDCYFFPNPLHCY 253 ANG153 SVAHVHGLLLLPESDALL 254 ANG154 GEYDCYFYPNPPHCYDRN 255 ANG156 LHVDCYFYPNPPHCYIDE 256 ANG157 QDHDCYFYPNPPHCYLEG 257 ANG158 HFSDCFFYPNPQHCYHMM 258 ANG159 PIEDCYFYPNPRHCYLQP 259 ANG160 ECCYCYNYDTWSHCCQKG 260 ANG161 VVLPSYDCYFYPNPPIICY 261 ANG163 VPWDCYFYPNPPHCYEDA 262 ANG164 FVSSAFDCYFYPNPAHCY 263 ANG165 ALVECYFYPNPLHCYNTS 264 ANG166 FWYSLDDCYFYPNPSHCY 265 ANG169 RSNLMTDCYYYPNPPHCY 266 ANG172 QLVDCLYYPNPPHCYESM 267 ANG174 WDVDCGFFPNPAYCYELG 268 ANG175 LQGRMIDCYSYPNPPHCY 269 ANG176 QRDPLAACYIYPNPPHCY 270 ANG179 ELWECYFYPNPSHCYTDR 271 ANG180 QDIDCYFYPNPPHCYHVT 272 ANG182 RLHDCYFYPNPPHCYLWQ 273 ANG183 SLTTFTDCYFYPNPPHCY 274 ANG186 SSGPDIDCYFYPNPPIICY 275 ANG187 DCYFYPNPLYCYEDWDYT 276 ANG188 IWLDCYFYPNPPHCSLNS 277 ANG189 QWNDCYFYPNPPHCYGRQ 278 ANG190 GHMDCYYYPNPSHCYQQL 279 ANG192 WLSQCYSYPNPPHCYTLF 280 ANG193 SSLHMLDCYYYPNPPHCY 281 ANG194 QSLSFLDCYFYPNPLHCY 282 ANG197 NYYYCYLSPNPPHCYLSF 283 ANG198 HFSDCYYFPNPPHCYESI 284 ANG199 EVLDCFFYPNPPHCYKAL 285 ANG201 RDTDCYFYPNPAIICYLGT 286 ANG203 KCPMEPDWVSCDEWMLQK 287 ANG210 LSLKCPLLPDWISCDEYY 288 ANG211 KCPLLPDWLSCLEWQLME 289 ANG215 KCPLLPDWVPCHEWLNVW 290 ANG217 AMSLCPLLPDWASCNDYL 291 ANG218 LCPRLPDWVSCHTWLERY 292 ANG219 LSTKCPLLPDWVSCHEWL 293 ANG220 LGWTELKCPLLPDWATCN 294 ANG222 KCPLLPDWVSCHEWNVWL 295 ANG223 KCPLLPDWISCAQWQRAV 296 ANG224 VGSLCPLLPDWASCHEWL 297 ANG225 RCPFLPDWLSCNEWLMIE 298 ANG228 WYRKCPLLPDWVSCHEWY 299 ANG229 TTLNCPLLPDWLTCQEFL 300 ANG231 KCPLLPDWASCESWLEVR 301 ANG232 HLDKCPLLPDWISCTDWL 302 ANG233 KCPELPDWVTCDEFQGMQ 303 ANG239 ATSKCPLLPDWVSCDEHL 304 ANG241 SSGKCPLLPDWKSCDEWL 305 ANG242 KCPLLPDWVSCHEWNIRE 306 ANG245 KCPLLPDWFSCIIEYQEMF 307 ANG246 KCPLLPDWVSCHTYLEES 308 ANG247 EPPKCPMLPDWVSCDTWL 309 ANG248 DVPHQLNCPLLPDWAACL 310 ANG249 ETSKCPLLPDWVSCYQWL 311 ANG250 MNVKCPLLPDWVSCLDYL 312 ANG252 MLMKCPLLPDWASCDVWL 313 ANG256 LMHECPLLPDWVSCHTFL 314 ANG257 KCPLLPDWVSCETYLNGD 315 ANG258 KCPLLPDWVSCDMYLERQ 316 ANG259 KCPLLPDWASCDQWNEWN 317 ANG263 YVSKCPFLPDWVSCHGWL 318 ANG266 KCPFLPDWVSCHDYSQKY 319 ANG267 FPPKCPLRPDWVSCHERH 320 ANG269 ECPLLPDWVSCIIEWNRLII 321 ANG270 SPVKCPFLPDWISCDLYL 322 ANG272 TAFKCPPLPDWVSCLDWL 323 ANG274 KCPLLPDWVSCQEWLSSK 324 ANG275 HYDKCPLLPDWVSCHEFL 325 ANG277 GLRKCPVLPDWVSCDNWL 326 ANG278 SGFKCPLLPDWVSCTDCT 327 ANG279 LASKCPLLPDWISCQTWL 328 ANG280 KCPLLPDWVSCDQWRLLE 329 ANG281 YFDKCPLLPDWLTCHEWM 330 ANG284 LPSKCPLLPDWTSCDEYL 331 ANG303 KSVSMKTCQDYPGWRYCQ 332 ANG335 VNVFLKTCQDYPGWRYCH 333 ANG339 PWPDCPVYRYCRHCHHKT 334 ANG355 TGSTAKYCQDYPGWRYCH 335 ANG358 LPMECYFYPNPEHCYLVP 336 ANG370 TYTKCMTYPNPQYCFKHS 337 ANG371 RFLECAWFPTPYFCHESE 338 ANG374 QYDPCEYYIPCVKWG 339 ANG375 GFHRCSTFPNPHYCYKTH 340 ANG376 RWSYCEETMIGYPYCFTRP 341 ANG377 AAFMLSCHSTDGQSWACMESH 342 ANG378 RSFECRWQPEHPICAKGV 343 ANG380 ILLRCAWYPAPPWCNIET 344 ANG381 PHVICHYWKHPWCFQNS 345 ANG382 VFYRCMKWPNPWWCYSQP 346 ANG383 GFQRCKTFPNPHYCYKTH 347 ANG384 DWSKCPKYPNPEWCYHPS 348 ANG385 TYTKCMTYPNPQYCFKHN 349 ANG387 SMWGCHMQPNYRECSLWI 350 ANG388 PHVICHYCPHPWCFLNS 351 ANG389 LELPCGKYPNMWFCYKVA 352 ANG390 KERPCVWWKQPWCFQAS 353 ANG391 QWFRCYEPPEPDWCRVRS 354 ANG393 EWAFCYSYPNPDYCHEAV 355 ANG394 TYRKCMTYPNPHYCFKHS 356 ANG395 RYSLCMTYPNPYQCYKSN 357 ANG396 PHVICHYWKHPWCFHNS 358 ANG397 PWFSCYYFPNPSHCMDVV 359 ANG398 LYDRCVCFPNLFWCEMTT 360 ANG399 RSLACEFHPQKYHVQCALPH 361 ANG400 NTLRCYSGVLVDAMVCFTSG 362 ANG403 QYDPCEYDIPCVKWG 363 ANG404 SWAQCYAYPNPDFCNRSG 364 ANG405 PYEKCMTYPNPWYCFLTD 365 ANG406 PYERCTKHPNPTYCWKNL 366 ANG407 MYAMCQWIIPSPEQCRSPS 367 ANG599 KCPLLPDWASCR 368 ANG637 TYLQIIECYFYPNPPHCY 369 ANG638 LTPSGSDCYFYPNPLHCY 370 ANG639 GAYDCYFYPNPPHCYGTT 371 ANG640 YLNHNYECYFFPNPTHCY 372 ANG641 GFLTFYDCYFFPNPPHCF 373 ANG642 TFIDCYFYPNPPHCYALW 374 ANG643 ASDILLDCYFFPNPPHCR 375 ANG644 LPLSWSDCYFYPNPPHCY 376 ANG645 ALLDCYFYPNPAHCYITM 377 ANG646 GMLDCYFYPNPPIICYLDR 378 ANG647 SGTECYPYPNPPHCYSSD 379 ANG648 SLEDCFFYPNPTLCYNNG 380 ANG649 AFSRFLDCYFYPNPQHCY 381 ANG650 GVYSFLECSFFPNPPYCY 382 ANG651 EEGSSSDCYFYPNPRHCY 383 ANG652 LTLLLDDCYFYPNPPHCY 384 ANG653 LFHSMTDCYFYPNPRHCY 385 ANG654 REQDCFFYPNPPHCYVDN 386 ANG655 ASLGLYDCYYFPNPPNCY 387 ANG657 RTGTLGDCYFYPNPRHCY 388 ANG658 SPLDCYFYPNPPHCYYAH 389 ANG659 IEGDCYFYPNPPHCYEQN 390 ANG660 FNYSMYDCYFYPNPAHCY 391 ANG661 LVLPLFDCYFYPNPPHCY 392 ANG662 FHFVLYDCYFYPNPPYCF 393 ANG663 HRADCYFFPNPMDCYQWR 394 ANG664 STNLALDCYFYPNPPHCY 395 ANG665 SPNDCYFYPNPPECYTHR 396 ANG666 IVITSLACYFYPNPLHCY 397 ANG667 GGVPMTDCYFYPNPRHCY 398 ANG668 DFRDCYSFPNPTQCYINT 399 ANG670 WPTDCFFFPNPPHCYHDG 400 ANG671 HILDCYFYPNPPHCYLVT 401 ANG672 DEADCYYYPNPPHCYSGH 402 ANG673 TLVSMMDCYYYPNPPHCY 403 ANG674 QSVRYMDCYFYPNPSHCY 404 ANG675 WYDPYYACYFYPNPTYCY 405 ANG676 LSGPFLDCYFYPNPTHCY 406 ANG677 VMLDCYFYPNPLDCYWLT 407 ANG679 SSLEHNDCYFYPNPMHCY 408 ANG680 RHVDCYFYPNPPHCYLVL 409 ANG681 IYPECYFYPNPKSCYSDQ 410 ANG682 PPTSLADCYFFPNPLHCY 411 ANG683 LLAACYFYPNPPLCFTST 412 ANG684 GEEDCYFYPNPPIICYSEL 413 ANG685 ECYFYPNPPHCYMEHRST 414 ANG686 TEIECYFYPNPPQCYAIH 415 ANG687 PRMDCYFYPNPPHCYNGQ 416 ANG688 MSLPHQNCYFFPNPTYCY 417 ANG689 YGDECYFFPNPPHCFSWD 418 ANG691 FFESSFDCYFFPNPNHCY 419 ANG692 MLVVPSDCYFYPNPSHCY 420 ANG693 LQVDSLDCYFYPNPPHCY 421 ANG694 QLMDCYFFPNPHHCFESK 422 ANG695 DCYFYPNPPHCYNERYEA 423 ANG696 SDMDCYFYPNPPIICFTPP 424 ANG697 GADTMQWNDCYFFPNPTHCY 425 ANG698 HIKDCYFFPNPPHCYKST 426 ANG699 PHQRHLECYFYPNPPECY 427 ANG700 SYLDFTDCYFYPNPPHCY 428 ANG701 APGYTQDCYWFPNPPHCY 429 ANG702 RFSSFHDCYFYPNPSHCY 430 ANG703 HSHHLTDCYYFPNPLHCY 431 ANG704 AQVDCYFFPNPTHCYFDR 432 ANG705 MGDDCYFYPNPPRCYVKP 433 ANG706 FLADCYLFPNPPHCYLDY 434 ANG707 LGEDCYWYPNPPHCYGML 435 ANG708 NDSVVSECYFFPNPPHCY 436 ANG709 DCYFYPNPPSCYEVQGAS 437 ANG710 GPWECFFYPNPLHCFKEP 438 ANG711 PSADFAECFFYPNPPQCY 439 ANG712 ASNDIHDCYFYPNPPHCY 440 ANG713 SDTDCYFYPNPPHCYWDT 441 ANG714 AFVHCYFFPNPQHCYHRL 442 ANG715 MIFDCYFFPNPRHCYHEG 443 ANG716 SYFLTWDCYTYPNPPHCY 444 ANG718 TGFECYFYPNPRIICYNDI 445 ANG719 LYPSLLDCYFYPNPTHCY 446 ANG721 MVDECYFYPNPPHCYSNT 447 ANG722 PLRSYMDCYWYPNPPHCY 448 ANG723 WLGRLSDCYFYPNPPHCY 449 ANG724 FHIDCYFYPNPPHCYTGA 450 ANG726 NFRECVFYPNPPHCYDFG 451 ANG727 KEIDCYFYPNPPHCYGKW 452 ANG728 TNQDCYFYPNPPHCFRDH 453 ANG730 SNAQCYFYPNPLHCYKNA 454 ANG731 DCYFYPNPPHCYTVEGGK 455 ANG732 QPMDCFFYPNPPHCYAEE 456 ANG733 IVSREYDCYFYPNPTHCY 457 ANG734 SMHDCYYYPNPTHCYRGT 458 ANG736 DETECYWFPSPAIICYKDS 459 ANG740 LGLDCYFYPNPPDCYHDA 460 ANG741 FDEDCYFYPNPPHCYLLS 461 ANG742 EHEACYFYPNPPHCYESS 462 ANG743 PSYNIFECFFFPNPPHCY 463 ANG744 SESDCYFYPNPPHCYAVV 464 ANG745 HPFDCYFYPNPPHCYTRS 465 ANG746 LILDCYFYPNPPHCYTST 466 ANG747 YEFRWTDCYFYPNPPHCF 467 ANG748 QSQNVVECYFYPNPPHCY 468 ANG749 YRLDLSECYYYPNPLHCY 469 ANG751 IPLDCYFYPNPPSCYIISY 470 ANG752 AASQTSDCYFYPNPPSCY 471 ANG755 THLDCYFYPNPPHCYSST 472 ANG756 NTNDPFDCFFYPNPPHCY 473 ANG757 IHKLDLDCYYYPNPPHCY 474 ANG762 FSHPFTDCFFYPNPQHCY 475 ANG763 TVYDCYFYPNPGHCYLQL 476 ANG765 MVFHELDCYFYPNPPHCY 477 ANG766 HSLLHWDCYFYPNPSHCY 478 ANG767 QLRECYWFPNPSHCYIES 479 ANG769 GLVDCYFYPNPPHCYWPH 480 ANG771 LFENSLGCYFFPNPPHCY 481 ANG773 FPPWFQDCFFYPNPPVCY 482 ANG774 HALECYFYPNPLHCYTQP 483 ANG775 DPVDCYFYPNPAHCYSTV 484 ANG776 IVVYSADCFFYPNPRHCY 485 ANG777 RMFDCYFYPNPPLCYNHI 486 ANG781 LWYTTVNCYPCPNPLHCY 487 ANG782 DYLSMLECYFYPNPSHCY 488 ANG783 FMTSGVECYFYPNPPHCY 489 ANG784 LFLNPYDCYFYPNPPQCY 490 ANG785 IIQMTQIECYFFPNPQIICY 491 ANG786 PWSPCYWYPNPRFCYEAS 492 ANG787 LLVDCYYYPNPTHCSKNS 493 ANG788 DCYFYPNPPHCYHDNEIE 494 ANG790 PLLPFYDCYFYPNPSHCY 495 ANG791 LVVDCYFYPNPPHCYDYS 496 ANG793 RPSMHMECYFYPNPPYCY 497 ANG795 GFLTSLDCYFYPNPPHCY 498 ANG797 EVPDCYYYPNPPHCYEAN 499 ANG798 RISSMVECYFYPNPPHCY 500 ANG799 PFSVITECYFYPNPPHCY 501 ANG800 PSADLLDCYFFPNPPHCS 502 ANG801 QQADHIDCYFYPNPPYCY 503 ANG802 STHDCFLYPNPPHCYWPQ 504 ANG803 VASWKYDCYFYPNPLIICY 505 ANG804 VDIDCYFYPNPPNCYNKA 506 ANG805 LLDRFLDCYFYPNPSHCS 507 ANG806 ECYFYPNPAHCYTGTNKE 508 ANG807 LPHPHLDCYFYPNPAFCY 509 ANG808 SYVDTTDCYFYPNPQHCY 510 ANG809 PPLDCYFYPNPPNCYLGQ 511 ANG810 WINDCYFYPNPSHCFVGD 512 ANG811 YGTDCYFFPNPRNCYKIN 513 ANG812 YSTDCYFYPNPPHCYGPH 514 ANG813 PFSSFFDCYFYPNPHHCY 515 ANG814 YVGIIMLDCYFYPNPPIICY 516 ANG815 SMYDCLYFPNPPHCYIQN 517 ANG816 FTQPMVDCYFYPNPPHCY 518 ANG817 FNSTLFDCFFYPNPMHCY 519 ANG820 RSLPIYECYFYPNPPHCY 520 ANG822 VSLYYSACYFYPNPPWCN 521 ANG823 SVLDCYFYPNPAHCYENV 522 ANG824 VSTDCYFYPNPPHCYSVL 523 ANG825 QGTHMLDCYFYPNPPNCY 524 ANG826 TPSDCFFYPNPPHCYLMN 525 ANG827 TWGVCYFYPNPPHCYEQL 526 ANG829 VEEACYFYPNPPHCYEAV 527 ANG832 DDSDCFFYPNPPRCYETY 528 ANG834 YYYSYTHCYYYPNPTHCY 529 ANG835 MTWFPNDCYFYPNPPYCY 530 ANG836 LQLECYYYPNPTHCYETR 531 ANG837 MGISFLDCYFFPNPQHCY 532 ANG838 PSPYYSDCAWYPNPPYCY 533 ANG840 SMSVYIDCFSYPNPPRCY 534 ANG841 DCYFYPNPTHCYLKGGFV 535 ANG844 DHSACYAFPNPSSCYNSR 536 ANG845 PLNACYFYPNPPHCYFLK 537 ANG846 LLSVMQDCYFYPNPSHCY 538 ANG847 PPPSSYECYFYPNPAHCY 539 ANG848 SHTDCYYYPNPPHCYLEN 540 ANG849 AGHECYFYPNPPHCYSEQ 541 ANG850 AGMNDFDCYFFPNPPHCF 542 ANG853 GSRDCYFYPNPKYCYIQD 543 ANG854 SDSFLRDCYFYPNPPHCY 544 ANG855 AWKDCFFYPNPSFCSYSS 545 ANG856 PSLYELDCYFYPNPPHCY 546 ANG857 YLHSFEDCYFYPNPAHCY 547 ANG858 LTIEHQDCYFYPNPPHCY 548 ANG861 YVMGGDDCYFYPNPPECY 549 ANG862 GNIACYFYPNPPHCYNVH 550 ANG863 SGMECYFFPNPPHCYDLK 551 ANG866 LGLRHYACYLYPNPPHCY 552 ANG867 ARSSQFECYFYPNPPHCY 553 ANG869 YPGPFTGCYFYPNPLQCY 554 ANG871 NERDCYFYPNPPHCYQNG 555 ANG872 LDSECFFYPNPPHCYNTL 556 ANG873 PYLDCAEYPNPLYCYESE 557 ANG874 IGYPFSDCYFYPNPPHCY 558 ANG875 WNFDCYFYPNPPLCYNME 559 ANG877 AAVECLFYPNPPHCYERA 560 ANG879 SNVDCYFYPNPPNCFVIID 561 ANG880 VLSFFQDCYFYPNPANCY 562 ANG881 SPPVFWECYFYPNPTHCY 563 ANG882 PNQSNYDCYFYPNPPHCY 564 ANG883 LAFDLSDCYFYPNPPHCS 565 ANG884 FNEECYFYPNPPHCYGEY 566 ANG885 LVVTAFDCYFYPNPPYCY 567 ANG886 SLNPSVDCYFYPNPAHCY 568 ANG887 ALHDCYFYPNPQHCYQA 569 ANG889 FSPGEVDCYFYPNPPHCK 570 ANG890 ADTDCYFYPNPPHCYDIT 571 ANG891 LSLELSECYFFPNPPHCY 572 ANG894 AVLEHFDCYFYPNPPHCY 573 ANG895 MLWDMSGCYFYPNPPHCY 574 ANG896 SKQDCYFYPNPSHCYLDQ 575 ANG897 YDLLLHDCYVFPNPPHCY 576 ANG898 ECYFYPNPPDCFYLIIQRR 577 ANG899 DHVECYFYPNPLHCYKDR 578 ANG901 PRPLVIDCYFFPNPPHCY 579 ANG902 DCYFYPNPPHCYIEHKPS 580 ANG903 SHFSFKDCYFYPNPAHCY 581 ANG904 SLRDCYFYPNPAHCRNNM 582 ANG906 YGNECYFFPNPPHCYHRD 583 ANG907 DTLDCYYFPNPPHCYSSQ 584 ANG909 QWNPHYHCFFYPNPPHCY 585 ANG911 TSLDCYFYPNPPHCYETP 586 ANG912 KWARGMECFFYPNPPDCY 587 ANG913 RLVDCYYYPNPLHCYDRS 588 ANG914 THKDCYFFPNPPHCYTKT 589 ANG915 PLKDCYFYPNPLHCYLVS 590 ANG916 APHILQDCYFYPNPRHCY 591 ANG917 LSILYPSCYFYPNPEHCQ 592 ANG918 ECYFYPNPPECYNVDDQA 593 ANG919 ECYFYPNPPHCYVLKQQI 594 ANG920 ISTRNTDCYFYPNPPHCY 595 ANG923 FYYDCFFYPNPPNCYNSF 596 ANG924 FSHLEQDCYFYPNPPHCY 597 ANG925 SPVDCYFYPNPPHCYDWD 598 ANG926 PYQLMTDCYFFPNPPHCY 599 ANG929 ESFQNMDCYFYPNPPYCY 600 ANG930 PRKSFHDCYFYPNPPHCY 601 ANG932 SWPNHMECYFFPNPPHCY 602 ANG934 KSVGNTDCYFFPNPPHCY 603 ANG935 TIYDCYIYPNPRHCYKLN 604 ANG936 ECYFYPNPPHCYHALTSK 605 ANG937 FYQSWVDCYFYPNPPHCS 606 ANG939 YALDLGGCYWFPNPPIICY 607 ANG940 SIFLSLDCYFFPNPSNSL 608 ANG942 GVYSFDQCYVFPNPPDCY 609 ANG943 VVLHYGDCYYYPNPPYCY 610 ANG944 RALDCYFYPNPQHCYQGT 611 ANG945 SPHECYFYPNPPHCYADR 612 ANG946 DVQIEFDCYFYPNPPYCY 613 ANG947 YPMNSHDCYFYPNPPQCY 614 ANG948 SPAHCSYCPFPPNSCFCY 615 ANG949 LRLDLEECYWYPNPPYCK 616 ANG951 GSALLSDCYFYPNPPHCY 617 ANG952 SVLALEDCYFYPNPHHCY 618 ANG954 PTAVLMECYFYPNPDYCY 619 ANG955 YGTECYFYPNPAHCYSNE 620 ANG956 HWYFCPNPPNPPQCHLKW

TABLE 11 VEGF binding MRDs SEQ ID: Ref. No. Peptide Sequence 629 VGF1 WCWVPGPIQCY 630 VGF2 CMWEMPWWFCQ 631 VGF3 TCTWVMPWWFCE 632 VGF4 ICLWQMPWWFCE 633 VGF6 GDWEWVWDWCF 634 VGF8 ECRWVMPWWFCE 635 VGF9 ACWKAMPWWFCT 636 VGF10 FCMVQVPWWLCK 637 VGF11 FYQWFEEQLM 638 VGF13 MCWLVPGPMICH 639 VGF14 FCMYKPLMFLCP 640 VGF15 HCWWLYTWDPCI 641 VGF16 HCYVYVPWWLCP 642 VGF17 MCWTPVPWWLCL 643 VGF18 TCWKPTPWWLCD 644 VGF19 SCWFKIPWWLCP 645 VGF20 YCQPMWWNMACW 646 VGF21 YCFWIPPEWYCV 647 VGF23 GCWEWRPHWLCV 648 VGF24 ECTWTMPWWFC 649 VGF25 TCWKPAPWWLCA 650 VGF27 TCWKPTPWWLCA 651 VGF29 KCNFNPWKAACG 652 VGF30 TCWKPAPWWLCD 653 VGF31 TSWKAAAWGLCA 654 VGF32 ACWKATPWWLCD 655 VGF34 TCWKATPWWLCA 656 VGF35 ACWKAAPWWLCD 657 VGF36 TCWKAAPWWLCD 658 VGF37 ACWKPAPWWLCD 659 VGF38 TCWKATPWWLCD 660 VGF39 TCWKAAPWWLCA 661 VGF40 ACWKAAPWWLCA 662 VGF47 WCAGFPPNYPCY 663 VGF48 WCNGFPPNYPCS 664 VGF49 WCNGFPPNYPCY 665 VGF53 WCNGFPPNYPCA 666 VGF57 GCVKEVPWWLCI 667 VGF59 GCVKQVPWWLCV 668 VGF60 GCVTQVPWWLCI 669 VGF62 ACVKEVPWWLCI 670 VGF63 GCVKQVPWWLCI 671 VGF65 ACVAEVPWWLCI 672 VGF67 GCVKAAPWWLCV 673 VGF69 GCVKEVPWWLCV 674 VGF70 GCVKPAPWWLCI 675 VGF71 GCVTEVPWWLCI 676 VGF72 GCVAQVPWWLCI 677 VGF76 ACVKEAPWWLCI 678 VGF82 GCVAEVPWWLCI 679 VGF83 ACVKQVPWWLCI 680 VGF85 GCVKPAPWWLCV 681 VGF95 GCVAEVPWWLCT 682 VGF99 GGVKQVASWLGT 683 VGF100 GAVTEAAWWLAI 684 VGF101 GGVEAAAWWVGI 685 VGF104 GCVKQVPWGLCI 686 VGF107 VEPNCDIHVMWEWECFERL 687 VGF131 WCAGFPPTSAGY 688 VGF140 TAWEAAASWLCA 689 VGF146 TCGKAAAAWLAA 690 VGF152 TGWKPTAWGVGD 691 VGF156 ASWKPAPWWLCA 692 VGF162 TSGKATPGGACD

TABLE 12 IGF1R binding MRDs SEQ ID: Ref. No. Peptide Sequence 698 IGF3 DSGFYMGLHK 699 IGF10 MDQFHWALLG 700 IGF21 LCQEFHELCF 701 IGF22 LCDEFVELCF 702 IGF23 LCEEFHELCF 703 IGF24 LCDEFKELCF 704 IGF25 LCAEFPELCF 705 IGF27 ICIIIFWQTCNGQAGQGGGSGGS KCDILEMMCN 706 IGF28 LCMEFPELCF 707 IGF29 LCMEFKELCF 708 IGF30 LCEEFPVLCF 709 IGF31 LCQEFIELCF 710 IGF32 LCLEFIIELCF 711 IGF33 LCQEFSELCF 712 IGF34 LCEEFAELCF 713 IGF35 LCFEFVELCF 714 IGF36 LCSEFQVLCF 715 IGF38 LCLEFVELCF 716 IGF39 LCWEFPVLCF 717 IGF40 LCEEFIELCF 718 IGF42 WCEISTTFCNGQAGQGGGGGG SQCQLLHMFCH 719 IGF43 LCMEFEQLCF 720 IGF44 LCLEFRELCF 721 IGF45 LCQEFAELCF 722 IGF46 LCQEFKELCF 723 IGF48 LCLEFEELCF 724 IGF49 LCYEFEELCF 725 IGF50 LCMEFQELCF 726 IGF51 LCIIEFEELCF 727 IGF52 LCYEFHELCF 728 IGF53 LCDEFQELCF 729 IGF54 WCQIQAFHCRGQAGQGGGSAG GGSSCNILQLMCS 730 IGF55 FECVLWNFVCK 731 IGF56 LCAEFPVLCF 732 IGF57 LCTEFKELCF 733 IGF59 LCQEFNELCF 734 IGF60 AMTLDEWIRMRGQAGGSWVD PAFWGDF 735 IGF61 FYGWFEEQIL 736 IGF64 FYEQLEKLVY 737 IGF66 FYEWFEEQLI 738 IGF67 FYEWFEAQIM 739 IGF68 FYDWFDMHVK 740 IGF69 SFYDMIELLIGQAGQGGGSGGG GGGSTIHFAQEEHP 741 IGF72 FYEWFEAQVM 742 IGF73 FYDWFAEQVI 743 IGF74 FYEWFTEQVN 744 IGF75 FYEWFIIEQVI 745 IGF76 QFYEWFEAQT 746 IGF77 FYEWFQLQMD 747 IGF78 VHWEGYQWVY 748 IGF79 FYETLFELAY 749 IGF82 FYEWFEAALI 750 IGF83 FYDWFDMQVQ 751 IGF84 FYEWFEPQVT 752 IGF85 EFYYWFFDQY 753 IGF91 FYEWFDMQVK 754 IGF92 ADFYQWFQNQV 755 IGF93 SFYAWFDEQV 756 IGF95 EVWEGYDWLY 757 IGF196 SHPLCTEFIELCFGFE 758 IGF253 VPFLCLEFSELCFTDP 759 IGF254 ADLLCAEFTELCFDVI 760 IGF255 AIIPLCLEFSELCFEAV 761 IGF256 PHPLCQEFEELCFETV 762 IGF257 ASQLCGEFPELCFDKV 763 IGF258 IPLLCFEFVELCFDLT 764 IGF259 DDALCKEFPELCFQNV 765 IGF260 IPYLCLEFPVLCFQDT 766 IGF261 PHILCLEFAELCFDHL 767 IGF263 VPALCLEFKELCFHDR 768 IGF264 TPALCLEFWELCFDEA 769 IGF265 VPPLCMEFTELCFHDP 770 IGF266 AHPLCVEFSELCFNED 771 IGF267 IPMLCWEFTELCFDLN 772 IGF268 VPQLCLEFQELCFGEH 773 IGF269 VPSLCLEFPELCFNEA 774 IGF270 PHPLCLEFIELCFGTH 775 IGF271 IPFLCLEFEELCFHLD 776 IGF272 EHPLCLEFPELCFSHP 777 IGF273 PHYLCLEFSELCFDER 778 IGF274 TPFLCLEFHELCFGEH 779 IGF275 PQLLCLEFAELCFWDI 780 IGF276 PHALCREFHELCFEKS 781 IGF277 RLPLCVEFSELCFDLR 782 IGF278 VPALCWEFPELCFHYQ 783 IGF279 TPSLCLEFSELCFADG 784 IGF280 VHWLCAEFEELCFDQT 785 IGF281 LHFLCLEFQELCFDSN 786 IGF282 VPPLCLEFPELCFTDS 787 IGF283 DHWLCIEFSELCFGKS 788 IGF284 EHPLCEEFKELCFGTN 789 IGF285 DWRLCQEFTELCFMDG 790 IGF286 YLSLCAEFTELCFIITE 791 IGF287 PPLLCFEFPELCFGYE 792 IGF288 DHKLCNEFPELCFHDV 793 IGF289 AHALCLEFLELCFEER 794 IGF290 IPFLCYEFQELCFGTL 795 IGF291 AELLCREFTELCFLRD 796 IGF292 APGLCIEFRELCFDEK 797 IGF293 YYALCAEFQELCFDEH 798 IGF294 VPLLCFEFPELCFGYQ 799 IGF295 VPSLCIEFSELCFDSP 800 IGF296 DYALCLEFRELCFGMN 801 IGF297 AIIRLCQEFVELCFGET 802 IGF298 PHFLCMEFPVLCFNDS 803 IGF299 IPQLCLEFSELCFGMT 804 IGF300 RTDLCMEFYELCFHSD 805 IGF301 KPPLCEEFHELCFDLT 806 IGF303 PHKLCLEFRELCFDEL 807 IGF304 LHFLCVEFVELCFDEA 808 IGF305 VPRLCYEFPELCFHKQ 809 IGF306 PHPLCLEFEELCFTER 810 IGF307 AARLCSEFPELCFHAS 811 IGF308 RHSLCIEFPELCFDEY 812 IGF309 AGRLCAEFHELCFGTN 813 IGF310 ASRLCMEFPELCFDRK 814 IGF311 HPALCVEFVELCFGHE 815 IGF312 IPPLCLEFQELCFGWD 816 IGF313 DDALCAEFNELCFEVT 817 IGF314 APPLCIEFEELCFGFL 818 IGF315 SHPLCLEFHELCFTDA 819 IGF316 IHPLCLEFTELCFHEV 820 IGF317 QEPLCVEFRELCFNED 821 IGF318 IPDLCREFEELCFGSK 822 IGF319 IIIPLCKEFPELCFDVD 823 IGF320 GPLLCAEFVELCFGDD 824 IGF321 YPPLCLEFPELCFWNH 825 IGF322 SHNLCLEFVELCFGDV 826 IGF323 PPLLCLEFHELCFHIE 827 IGF324 AHVLCLEFHELCFDMG 828 IGF325 RPLLCYEFSELCFDIT 829 IGF326 HPRLCLEFSELCFDEM 830 IGF327 IPALCLEFSELCFSTS 831 IGF328 APSLCLEFIELCFDNM 832 IGF329 PHPLCREFPVLCFDDT 833 IGF330 VHPLCLEFAELCFVNY 834 IGF331 VPSLCMEFPELCFTTE 835 IGF332 VPLLCLEFAELCFHVE 836 IGF334 AMRLCNEFIIELCFGVV 837 IGF335 EERLCKEFPELCFMMT 838 IGF336 TPSLCREFTELCFDSD 839 IGF337 VHPLCMEFTELCFHLR 840 IGF338 AGALCKEFPILCFTME 841 IGF339 EHPLCREFSELCFGMG 842 IGF340 SSSLCLEFSELCFHDD 843 IGF341 ALRLCNEFPELCFGGT 844 IGF343 IPLLCLEFPVLCFENE 845 IGF344 APLLCLEFPELCFGWD 846 IGF345 PALLCQEFAELCFDMP 847 IGF346 YLIILCREFTELCFDEG 848 IGF347 AHWLCLEFKELCFGND 849 IGF348 VPSLCFEFEELCFDWS 850 IGF349 VPSLCLEFQELCFTTE 851 IGF350 PHPLCLEFPELCFGAE 852 IGF351 DSRLCAEFPELCFEDV 853 IGF352 AATLCLEFSELCFDMS 854 IGF353 IPHLCKEFPELCFEQR 855 IGF354 SYLLCREFSELCFDKT 856 IGF355 VPYLCLEFAELCFQVD 857 IGF356 VPPLCVEFSELCFDAP 858 IGF357 MELLCFEFSELCFGSH 859 IGF358 TVALCKEFPELCFDIV 860 IGF359 DPYLCLEFEELCFRFV 861 IGF360 TPALCLEFAELCFEDS 862 IGF361 RHPLCVEFPELCFVDY 863 IGF362 VHPLCIEFPALCFDTR 864 IGF363 YLALCYEFRELCFGDQ 865 IGF364 APLLCLEFPELCFEDI 866 IGF365 VHYLCLEFQELCFDEL 867 IGF366 RPSLCLEFRELCFDTL 868 IGF367 APSLCWEFKELCFDSE 869 IGF369 AHALCLEFSELCFDVT 870 IGF370 TQYLCIEFAELCFDTI 871 IGF371 AGALCREFAELCFHNH 872 IGF372 TPPLCFEFPELCFHLS 873 IGF373 SERLCLEFAELCFGHG 874 IGF374 AHWLCLEFPELCFDKL 875 IGF375 DLHLCREFSELCFSSP 876 IGF376 TPSLCLEFPELCFHGT 877 IGF377 APSLCLEFRELCFEDY 878 IGF378 VPSECLEFADLCFHDI 879 IGF379 MPALCLEFRELCFEDP 880 IGF380 YMRLCYEFSELCFHAS 881 IGF381 APSLCTEFRELCFDII 882 IGF382 VPPLCLEFAELCFYAD 883 IGF384 TPSLCVEFRELCFDDL 884 IGF385 QEALCIEFHELCFEEY 885 IGF386 ADRLCREFSELCFDVV 886 IGF387 AHPLCLEFPVLCFDDE 887 IGF388 RPVLCIEFPELCFNET 888 IGF389 DVKLCQEFPELCFESY 889 IGF390 VPALCFEFAELCFHIE 890 IGF391 AYALCHEFQELCFETT 891 IGF392 AALLCHEFTELCFLEA 892 IGF393 QHLLCIEFNELCFNDV 893 IGF394 IPPLCLEFWELCFDAP 894 IGF395 AHRLCMEFAELCFGNA 895 IGF396 RPSLCQEFVELCFEEA 896 IGF397 PHRLCIEFPELCFDVV 897 IGF398 AQALCLEFMELCFDVR 898 IGF399 ADALCAEFKELCFNIK 899 IGF400 GHKLCTEFIELCFDVN 900 IGF401 DEQLCLEFVELCFEQT 901 IGF402 VYALCREFSELCFYGN 902 IGF404 AGALCAEFPELCFGGT 903 IGF405 PPSLCLEFPELCFHRS 904 IGF406 IPALCIEFSELCFWPS 905 IGF407 VQPLCLEFSELCFDSA 906 IGF408 EHILCQEFSELCFNRD 907 IGF409 VPPLCLEFTELCFIID 908 IGF410 IPPLCLEFWELCFDER 909 IGF411 PHPLCREFPDLCFDHS 910 IGF412 PHWLCEEFEELCFEAI 911 IGF413 DRLLCSEFYELCFDDN 912 IGF414 PAALCLEFSELCFTQQ 913 IGF416 YSSLCLEFSELCFDQM 914 IGF417 RPALCGEFKELCFDGA 915 IGF419 YNALCNEFIELCFHVN 916 IGF420 PHWLCLEFAELCFGSG 917 IGF421 APPLCLEFRELCFGEQ 918 IGF423 TPQLCLEFTDLCFEDT 919 IGF424 VPHLCIEFAELCFEGT 920 IGF425 RMDLCREFTELCFTDS 921 IGF426 AHQLCIEFAELCFNDG 922 IGF427 PPLLCLEFPVLCFDSP 923 IGF428 PHALCLEFSELCFWSL 924 IGF429 VHQLCMEFEELCFGGA 925 IGF430 PHALCLEFHELCFDSN 926 IGF431 YRALCFEFAELCFDEN 927 IGF485 EFYQGLERLV 928 IGF498 YFYWFQEMVM 929 IGF500 FYQWFEKQLM 930 IGF503 QEFYTMWFALQI 931 IGF504 ECDFYCGIYTL 932 IGF507 LCREFQELCF 933 IGF511 GFYEWFEMQL 934 IGF512 FYEWFQAQLT 935 IGF514 GFYEWFEMQM 936 IGF515 YLSPDDMCYEFPELCF 937 IGF516 KTSQDMLCWEFEELCF 938 IGF517 QTTPHILCLEFPELCF 939 IGF518 LTSWELLCFEFEELCF 940 IGF519 EVSPEDLCQEFVELCF 941 IGF520 WYVDPPLCFEFPELCF 942 IGF521 LCAEFAELCFEDTVRY 943 IGF522 PGLEIPQLCKEFPELCFTMDDDF 944 IGF523 LCAEFRELCFHLPAQA 945 IGF524 MRVESLQLCKEFPELCFFSHGWT 946 IGF526 RLTPQDLCFEFSELCF 947 IGF527 LSSYDVLCQEFSELCF 948 IGF532 GPMEVHDLCKEFSELCF 949 IGF533 LRPESIALCKEFPELCFEGWKSA 950 IGF534 QSLDIPGLCKEFPELCFEMTGMR 951 IGF535 LCSEFIELCFDQVPLR 952 IGF536 TSSALKLCQEFPELCF 953 IGF537 LSSYMLLCMEFSELCF 954 IGF538 YVIPHPLCLEFSELCF 955 IGF539 DPSVPALCHEFPELCF 956 IGF540 QGWTHPLCLEFQELCF 957 IGF541 FALVPSLCLEFNELCF 958 IGF542 MSSNSIKLCKEFPELCFHIHGKD 959 IGF543 LCLEFRELCFDLSEPH 960 IGF544 MRLESLELCKEFPELCFSTDLTL 961 IGF545 TMLEIPQLCKEFPELCFHDTTVW 962 IGF546 LCQEFKELCFEAEFQA 963 IGF547 KVTPFMLCQEFPELCF 964 IGF548 APGEHWLCLEFQELCF 965 IGF549 VFQMIIPLCLEFPELCF 966 IGF551 YEPPHSLCLEFVELCF 967 IGF552 PPIMHPLCLEFAELCF 968 IGF553 LCAEFIELCFDLEPGI 969 IGF554 LSPEFPDLCLYFGLDC 970 IGF555 GSLSVPKLCKEFPELCFDKKMSG 971 IGF556 QRFEPDDLCKEFPELCFNLNMNV 972 IGF557 LCLEFAELCFGWDDNS 973 IGF559 RIYPSELCLEFAELCF 974 IGF560 QSYEHWLCQEFHELCF 975 IGF561 SSPPHWLCLEFEELCF 976 IGF562 RMSEMDLCAEFFELCF 977 IGF563 LCAEFHELCFDISNQE 978 IGF564 LCFEFAELCFDTEFYN 979 IGF565 PRLCKEFPELCFEVPVIGNEEHY SL 980 IGF566 QGSKVLELCKEFPELCFDRANRL 981 IGF567 TLSPHPLCVEFPELCF 982 IGF568 SEWYHPLCVEFSELCF 983 IGF569 SAGEIIWLCVEFQELCF 984 IGF570 LSPAELLCLDFPELCF 985 IGF571 LTPAERLCDEFPVLCF 986 IGF572 LCKEFPELCFDERDKK 987 IGF573 LTSADLLCSEFRELCF 988 IGF574 RITPNDLCQEFQELCF 989 IGF575 FAPPHPLCIEFQELCF 990 IGF577 VGKEVFRLCKEFPELCFYPAISQ 991 IGF578 LRFESPQLCKEFPELCFFESNGW 992 IGF579 YRAERPELCKEFPELCFFENDWW 993 IGF580 LCREFIELCFDIETEV 994 IGF581 RTVVPPLCLEFPELCF 995 IGF582 LYTPQSMCAEFTELCF 996 IGF583 GLVQHNLCLEFVELCF 997 IGF584 IDRPPDLCKEFPELCF 998 IGF585 VFLDAPELCKEFSELCFGYSDSL 999 IGF586 RFSESPPLCKEFPELCFWSPPFP 1000 IGF589 RDFTYDTDWEGYSWLY 1001 IGF592 IPGLCLEFPELCFHQW 1002 IGF596 EPWEGYSWLY 1003 IGF600 PLWEGYSWLYSQEAGN 1004 IGF602 FASGEYPPWEGYRWLY 1005 IGF603 WGDDHWEGYDWLYAQT 1006 IGF604 RDEAEWEGYNWLYSQM 1007 IGF605 DTWEGYLWLYAQTADA 1008 IGF607 YTRSLGDPWEGYSWLY 1009 IGF611 SAWEGYAWFDLQVESG 1010 IGF612 YSIQDHGAWPGYDWLY 1011 IGF613 QWGHKSEQWEGYSWLY 1012 IGF614 QQPNTWEGYSWLYSQV 1013 IGF616 DLWEGYLWFDSQVLGD 1014 IGF617 IPLLCLEFPVLCFENE 1015 IGF618 SYDYSQGEWEGYQWLY 1016 IGF619 HDSALMAEWEGYNWLY 1017 IGF623 QPWEGYKWLYEAA 1018 IGF625 PAASAWEGYKWLYDQV 1019 IGF630 LSTDWSGDWEGYAWLY 1020 IGF633 LLASEWEGYEWLYGQI 1021 IGF1270 ALPECQDLMFYECLFTL 1022 IGF1271 FYNTCQFVGMEFYQCLDWL 1023 IGF1272 RDPVCETKDVFYQMLCTVAF 1024 IGF1273 RSLVCSEEEPFYLALCRLTH 1025 IGF1275 SVLGCDDVGFYQCLEFL 1026 IGF1276 LEHLCQTRDGFYELMCTLAL 1027 IGF1277 AARWCDMQQHSFYECLQVL 1028 IGF1278 NALGCQEEWETLDQCLMRL 1029 IGF1279 AVPKCPGWDFYTALECLSRT 1030 IGF1280 EPRLCHNYPDFYQCMSFL 1031 IGF1281 ASQTCIGMDFYSGLECLLGW 1032 IGF1282 WVEECHNLHWDLKHCLMHQ 1033 IGF1283 SVPSCIDYYGDWFLCLAAP 1034 IGF1284 NNTLCRGVPTFEECVFIL 1035 IGF1285 SNGSCHIVDFYGWFQCQTSA 1036 IGF1286 LTEPCSQHSMFYSMLCELAF 1037 IGF1287 RDRVCHNKTFFHCLFSIFPDG 1038 IGF1290 YGLGCSQMDFYQCLDML 1039 IGF1291 DVSHCHIMFWDFQQCLEYL 1040 IGF1292 NPATCYQKSYQFEQCLMYL 1041 IGF1328 VDVLCHHVDFYECMDRL 1042 IGF1329 WLSQCEAKEIDFYMCLAGQAG 1043 IGF1330 GPKECIGVPNFDLCMAML 1044 IGF1331 LEDWCLHLDFYQCLEAV 1045 IGF1332 TMPTCEKLNFYECMNLL 1046 IGF1333 SLSICTYADFYECMDLL 1047 IGF1334 YIRECHELQNFTEGIHCLVTR 1048 IGF1335 TFKDCAHIPGIDFCLWRL 1049 IGF1336 SWDPCFESDEFIIMKLCVLMD 1050 IGF1337 LLSECYDKDFYECLGLE 1051 IGF1338 YQDMCEDMSFYECLEVL 1052 IGF1339 AAANCHWPDEALQELCRYFA 1053 IGF1340 TGICENPDEFYRQLCLLVE 1054 IGF1341 WEHHCRRSHLDFYGCLSSI 1055 IGF1347 AAGSPLDDCYGYTGFYQGMCL LEL 1056 IGF1348 PSYHCSNMDYTFYECLMYL 1057 IGF1349 EQSYCSQNDMFYDWFCALVS 1058 IGF1354 GEEVCWVQEPFYSTLCILAF 1059 IGF1355 NPFWCSWGHQFDNECDSMV 1060 IGF1356 WASTCQQTFYSMNRCLSQW 1061 IGF1360 LMELCAMDEDYYLWFCRAVE 1062 IGF1364 SAVQCDLRLMCWQLRGPSRP 1063 IGF1365 PSEICITEGLGFYQCLLKL 1064 IGF1366 WVDKCHQEMMEFHECLKYV 1065 IGF1370 ARCECWESQFYWDLCSLAL 1066 IGF1371 CTATCLEDQGFYWNMCKLVF 1067 IGF1373 AYDCRKHHMNFYECLTYL 1068 IGF1376 VGQMCSMGLELDRMKCNPCY 1069 IGF1378 LVDICQRQELDFYSCLRSV 1070 IGF1379 TGSDCYFYKHLYSLCVEVE 1071 IGF1380 ESGDCPWLGHVSFEECLMWL 1072 IGF1383 LHSVCFEAPEWQQCLMQR 1073 IGF1386 PGVSAHALCYEFPVLCFYDVG 1074 IGF1387 AGVSADALCYEFPVLCFYDVG 1075 IGF1390 DGESYFYVCLEHLLNVPGYHCV MTNG 1076 IGF1392 SYGTRWNPSFESNQFHMKLCVL MDGK 1077 IGF1395 PGVSADALCYEFPVLCFYDV 1078 IGF1396 HALCYEFPVLCFYDV 1079 IGF1401 TSRRCEEYQDPLKEVCRYFL 1080 IGF1405 AHALCYEFPVLCFYDVG 1081 IGF1406 AHALCYEFPVLCFYDV 1082 IGF1408 DFYICLEHLLNVPGYHCVMTNG 1083 IGF1409 DALCYEFPVLCFYDV 1084 IGF1411 NLMECIIALDFNQGMQCLQAY 1085 IGF1413 NLMECHALDFYQGMQCLQPYR HP 1086 IGF1414 DLMECHGLDFYKGMECLQAYG 1087 IGF1421 AHALCYEFPVLCFYDV 1088 IGF1422 NLMECHALDFNQGMQCVQAY 1089 IGF1430 AIIALCYEFPVLCFYDVG 1090 IGF1432 AYVCSNLNHCPALDFNQGMQC LQAYG 1091 IGF1437 VTEARTNDCNTTTFYHWFDCQ LGTG 1092 IGF1443 NLMHCHGLDFYQGMQCLQAY 1093 IGF1445 EAECRYPDTDLMWLCTYFSG 1094 IGF1446 DALCYEFPVLCFYDVG 1095 IGF1895 MAVGCTWVESCHWA 1096 IGF1896 ALPECQDLMFYECLFT 1097 IGF1897 FYNTCQFVGMEFYQCLDW 1098 IGF1899 SVLGCDDVGFYQCLEF 1099 IGF1900 AARWCDMQQHSFYECLQV 1100 IGF1904 EPRLCHNYPDFYQCMSF 1101 IGF1905 ASQTCIGMDFYSGLECLLG 1102 IGF1906 SNGSCHIVDFYGWFQCQTS 1103 IGF1987 ALAECIFEPTFEDCMWIL 1104 IGF1988 VDMQCQVHGMTFYECLMRL 1105 IGF1989 TLPECLYAPTFEICVEYL 1106 IGF1990 WEERCVDSDFYTQLCMLAW 1107 IGF1991 GTVLCPEDMDFYECMTFI 1108 IGF1992 REELCSVSPFDYHCRLFS 1109 IGF1993 LSYTCQHFDLPWELCLSLH 1110 IGF1994 VISECEGLTFYQCMASL 1111 IGF1995 WEEWCVDSDFYTQLCMLAW 1112 IGF1996 REPLCIQDDPIMYLHCYFSV 1113 IGF1997 MSYTCEDFDLPWELCLSLH 1114 IGF1998 DDLDCKCLTFYECMAAL 1115 IGF1999 WSNVCQARNMAFQECLEYL 1116 IGF2000 EDLGCLYLTFNQCMMWL 1117 IGF2001 FAHECSFHKLNFYQCLSFE 1118 IGF2002 LMDFCYVYPNICGCSN 1119 IGF2004 SRFVCQHDWGFYECLSML 1120 IGF2005 ASHICHLIPTIEQCLFSL 1121 IGF2006 VISECEGLTFYECMAAL 1122 IGF2009 VDMLCQVHGMTFYECLMLL 1123 IGF2010 WEEWCVDSDFYTQLCLLAW 1124 IGF2011 WDRWCVTEELPEQLCFLDG 1125 IGF2012 MSYTCEDFDLPCELCLSLH 1126 IGF2013 GLDSCQQAYFYQFFCEVFR 1127 IGF2014 VDMQCQVHGMTFYQCLMRL 1128 IGF2015 EDLGCMYLTFYECMIWL 1129 IGF2016 VSALCEDLEFYECILAL 1130 IGF2017 DVRECEGLTFYECMAAL 1131 IGF2019 TAWCHAAGLTFTQCLLWL 1132 IGF2020 CEEWCVDSDFYTQLCMLAW 1133 IGF2021 GISECEGLTFYECMAAL 1134 IGF2022 EYKVCTSNDTFHWGICALLK 1135 IGF2023 EDLGCMYLTFDECMMWL 1136 IGF2025 WKEWCVDSDFYTQLCMLAW 1137 IGF2026 FGQLCDQQVETMIECFERL 1138 IGF2027 DLFDCQGLTFYQCLAAL 1139 IGF2029 LQQICDPEIIVTFYQCMTLL 1140 IGF2030 QSLTCNDFTDWQSLCRC 1141 IGF2031 PREACHDYDVVLHQLCMYFV 1142 IGF2033 EDLGCVCLTFYECMIAL 1143 IGF2034 IPGLCLE FPELCFHQW 1144 IGF2035 LCWEGYKWFYDQIKPD 1145 IGF2036 GFHHAYY KYRGQAGQGS ATGGSSRITP SDLCKEFPEL CF 1146 IGF2038 RCLANSY HCSGQAGQGS ATGGSSRITP SDLCKEFPEL CF 1147 IGF2040 GSYCAPDWHN CFGQAGQGSR ITPSDLCKEF PELCF 1148 IGF2041 IPLLCLE FPVLCFENE 1149 IGF2042 SLEHLCQTRDGFYELMCTLA L 1150 IGF2043 SKDPVCKTKHQFYQMLCTLA F

TABLE 13 IL6 binding MRDs SEQ ID: Ref. No. Peptide Sequence 1158 ILB59 QRMEKFMHGS 1159 ILB60 MLDWVGRRPF 1160 ILB65 YFWHSLKTMM 1161 ILB69 DEIPLWEFQF 1162 ILB71 FQESWSTWVEGQAG 1163 ILB73 VWTAFIQMNS 1164 ILB75 ECTHPVFVRPCWGQAG 1165 ILB76 MWMYGWWWAG 1166 ILB77 FCIYYHNHMIWGQACQAGRCG SYCWWQDYAYKVGQAG 1167 ILB82 ICVASICGLVC 1168 ILB84 RWHYWHQHWWGQAG 1169 ILB87 VCKWKQEYCKGQAG 1170 ILB88 WYIIWMKEIIMWGQAG 1171 ILB89 HHHKMWPQNKSADSHAYTMM 1172 ILB91 WPSWTYWYHF 1173 ILB92 PWTYNWPWFM 1174 ILB94 IWHVFYMVQAGQAG 1175 ILB96 RYMHMVWQHI 1176 ILB98 PMTMWWFFWKGQAG 1177 ILB99 HSFVYMHWFL 1178 ILB100 WPMITWLGWYGQAG 1179 ILB101 YMFHWPLFWLGQAG 1180 ILB102 MQGWLWAKWW 1181 ILB104 IYAWWKWHWFGQAG 1182 ILB105 TMQPHHIYGYGQAG 1183 ILB106 SLFMPQHNVHGQAG 1184 ILB107 YWPSLEWDWS 1185 ILB114 GCWWVATECWGQAG 1186 ILB122 GCTWLIDICTGQAG 1187 ILB128 HQLQPYIQDL 1188 ILB130 WCLAGHVYCQGQAG 1189 ILB135 GVLKSGFRSLRCMARP 1190 ILB139 PCPGFPPPMLCMGQAG 1191 ILB140 PCMRDWWMCGGQAG 1192 ILB142 KCTHLPFQRNCSGQAG 1193 ILB143 FCHHWRPVCLGQAG 1194 ILB146 ACMLKTHRCKGPGR 1195 ILB148 RCSIWKVYAMCM 1196 ILB151 ECLWTSLECEGQAG 1197 ILB158 VCIPMATMCIGQAG 1198 ILB226 SVSWCFWDPLVMQCKDHT 1199 ILB228 VHFNCAWDPLMMRCNYDL 1200 ILB229 RMSTCLWDPLLMNCKSLW 1201 ILB230 TSEPCFWDPLLMTCDYGS 1202 ILB231 QNVNCLWDPLRMNCVEYK 1203 ILB232 SYIGYTYCPWDPLTMRCV 1204 ILB233 LRHFVQYCPWDPLLMACI 1205 ILB234 FTIGGPICGWDPLLMRCV 1206 ILB236 LLCSENWCRWDPLMMSCR 1207 ILB237 FSQMCGWDPLLMMCRSVN 1208 ILB238 VGSDCMWDPLMMNCRYSY 1209 ILB239 LSLLCHWDPLIMRCSYDS 1210 ILB240 YIPPCHWDPLLMDCAQIQ 1211 ILB241 MMMECHWDPLLMRCQNPK 1212 ILB242 LPSSCYWDPLLMECSHRN 1213 ILB243 LVPPLFACFWDPLLMNCS 1214 ILB244 WSYFNRSCAWDPLLMKCA 1215 ILB245 SSFTVVSCMWDPLLMSCM 1216 ILB246 LLTKYRTCHWDPLLMLCR 1217 ILB247 MVFWDEFCSWDPLLMACR 1218 ILB248 NQLSSIMCHWDPLLMTCQ 1219 ILB249 TFTSCSWDPLLMDCTDFV 1220 ILB250 MVFYCYWDPLLMLCHDQT 1221 ILB252 QVSLCFWDPLMMTCSVPF 1222 ILB253 ISMPCSWDPLLMNCAKWP 1223 ILB254 FEAWCVWDPLLMNCVTYT 1224 ILB255 NRYERFMCFWDPLLMMCS 1225 ILB256 VIMNQFFCFWDPLLMRCM 1226 ILB257 FSSAYSGCYWDPLLMECI 1227 ILB258 WSSGLYGCYWDPLLMLCK 1228 ILB259 RSSADWSCLWDPLLMQCM 1229 ILB260 ITSLERSCMWDPLLMLCS 1230 ILB261 MHGYCTWDPLLMRCSNYI 1231 ILB262 STVGCFWDPLLMDCRSMV 1232 ILB263 WLSTCLWDPLLMQCIESE 1233 ILB264 SGVSCFWDPLRMICYILP 1234 ILB265 YLSSCLWDPLLMRCTKLM 1235 ILB266 APQQCNWDPLLMNCVKYV 1236 ILB267 SYVTFLSCHWDPLRMQCI 1237 ILB268 FSSVTSACAWDPLIMRCI 1238 ILB269 ALALVASVTGTRYQCRV 1239 ILB270 TKNVTYECMWDPLLMVCT 1240 ILB271 PFYEYTCCPWDPLLMNCL 1241 ILB272 KDYFHQYCIWDPLTMNCA 1242 ILB273 HYTNCVWDPLLMRCYEFA 1243 ILB274 AVGWCPWDPLLMNCMGRN 1244 ILB275 VASVCLWDPLLMQCWSHY 1245 ILB276 DGMICLWDPIRMDCYYTE 1246 ILB277 DFTFCAWDPLLMACNDQF 1247 ILB278 DWEECVWDPLLMMCGISY 1248 ILB279 QRNYSEECHWDPLLMLCQ 1249 ILB280 ALVNRVECSWDPLLMKCS 1250 ILB281 NLDVTPYCYWDPLRMVCY 1251 ILB282 PRFLIYFCNWDPLLMMCK 1252 ILB283 YYPITLDCIIWDPLLMRCL 1253 ILB284 YIEINHPCHWDPLLMDCT 1254 ILB285 LVDVCSWDPLLMNCRLAN 1255 ILB286 FDTFCFWDPLLMDCAAPT 1256 ILB287 QSNDCAWDPLLMQCLNSL 1257 ILB288 MTSHCLWDPLLMVCVNHE 1258 ILB289 HFTFCAWDPLLMQCGIDS 1259 ILB290 KSTALITCIWDPLLMQCR 1260 ILB291 YTNNMETCAWDPLLMMCY 1261 ILB292 FSGKQMMCAWDPLMMQCA 1262 ILB293 LHLGEWDCYWDPLLMICR 1263 ILB294 WLFNGVDCLWDPLLMDCI 1264 ILB295 FHESLLPCFWDPLLMTCQ 1265 ILB296 LYMICTWDPLLMDCREDR 1266 ILB297 LNLLCAWDPLLMSCIETL 1267 ILB298 WEHGCMWDPLLMQCINRS 1268 ILB299 GSTCCVWDPLLMKCMSPV 1269 ILB300 GLYNCTWDPLLMRCSESI 1270 ILB302 DNWSISSCYWDPLLMNCP 1271 ILB303 WFLFPWTCPWDPLLMECI 1272 ILB304 SVQYETPCYWDPLRMQCQ 1273 ILB305 LDFSFFDCSWDPLLMRCS 1274 ILB306 FRMKKVDCCWDPLLMNCR 1275 ILB307 NAPVWVECMWDPLLMSCV 1276 ILB308 GYAWCFWDPLLMKCTWQV 1277 ILB309 DDFLCSWDPLLMDCMVYP 1278 ILB310 FEDSCAWDPLLMACSNPI 1279 ILB311 LAGSVSGTLLQQTVQRT 1280 ILB312 LVSFCAWDPLLMNCMWPM 1281 ILB313 LYGECAWDPLLMRCQPYY 1282 ILB314 GSKRFGICFWDPLLMDCA 1283 ILB315 YVAVGPSCLWDPLLMMCA 1284 ILB316 GFGSSWFCSWDPLLMQCQ 1285 ILB317 SISRAFSCVWDPLLMNCS 1286 ILB319 MTQSLSFCHWDPLLMYCV 1287 ILB320 LVPYSWYCVWDPLLMDCV 1288 ILB321 SDNLYFDCHWDPLLMMCR 1289 ILB323 LFTMCHWDPLLMDCATVP 1290 ILB324 FCSWDPMLMDCSTITFDM 1291 ILB329 TQMYCWWDPLLMNCTEMS 1292 ILB330 LTGYCYWDPLLMNCLEQG 1293 ILB331 NHPMCWWDPLMMDCMYFE 1294 ILB332 PDTLCIWDPLLMQCRWMP 1295 ILB333 VNVSFWGCVWDPLLMVCS 1296 ILB334 KAVLSNYCYWDPLLMICS 1297 ILB335 LGRLVPYCRWDPLSMVCL 1298 ILB337 RWKIDVICKVEILQRRCT 1299 ILB339 SHIACGWDPLMMMCTQEM 1300 ILB340 ICWWDPLLMDCISTVEPY 1301 ILB341 MFLSVVDCFWDPLLMECV 1302 ILB342 HCYWDPLLMTCYDSGPWV 1303 ILB343 IFEYCVWDPLLLRCSLNS 1304 ILB344 FVDDCVWDPLMMDCHRYS 1305 ILB347 KLALLDDCIWDPLLMSCR 1306 ILB348 WTNWCGWVLPTMLCMYWI 1307 ILB349 DWSTLYFCSWDPLLMLCS 1308 ILB350 GQNHCFWDPLMMICLNGH 1309 ILB351 TLLTCVWDPLLMQCRDLE 1310 ILB352 YVSPCVWDPLLMLCNESW 1311 ILB353 LCFWDPLYMECSTHLYID 1312 ILB357 LLAWCFWDPLLMDCVWDS 1313 ILB358 YHYLSVNCHWDPLLMECT 1314 ILB359 FHHFCFWDPLLMGCMNYN 1315 ILB360 FCVWDPLLMLCQWLEDSS 1316 ILB361 FNTQSTDCWWDPLLMLCA 1317 ILB362 VQLTCFWDPLMMWCSEQS 1318 ILB365 LRAICIWDPLMMLCGTMG 1319 ILB367 ISFPHFICPWDPLLMQCS 1320 ILB368 RSFVCNWDPLMMTCTSDL 1321 ILB369 PLWSCFWDPLLMDCQWTL 1322 ILB370 SHSSNMGCYWDPLMMMCH 1323 ILB371 RVYECYWDPLIMDCTWDL 1324 ILB377 FSVNYTPCVWDPLLMRCS 1325 ILB378 LVALPNSCFWDPLLMTCR 1326 ILB379 FWVGCPWDPLLMKCTSYN 1327 ILB380 KLALMSSCLWDPLLMDCM 1328 ILB381 HCYWDPLLMDCKYLILEG 1329 ILB382 GSIICWWDPLLMSCADLK 1330 ILB383 QLFTCIIWDPLLMDCTWDS 1331 ILB387 WLEQCVWDPLLMSCTSYV 1332 ILB388 SSPGIDACWWDPLLMDCA 1333 ILB389 LHFVCFWDPLIMDCSPQM 1334 ILB390 SYAYCYWDPLLMECADPM 1335 ILB393 VWLVFWPCFLDPISSICN 1336 ILB395 SLLECFWDPLMMNCSNGL 1337 ILB397 SDDWTMECYWDPLLMTCI 1338 ILB398 ALMFCFWDPLLMRCNANV 1339 ILB399 NRLMCFWDPLLMVCQSDL 1340 ILB402 LMQRPVHCVWDPLLMNCR 1341 ILB403 WQCYYRLCMGYPLGILCS 1342 ILB404 FRFATFFCYWDPLLMSCR 1343 ILB405 LTFLFYRCSWESVTPGCI 1344 ILB406 LLLSTYWCSWDPLLMDCN 1345 ILB407 SLWLTVLCPWDPLLMACI 1346 ILB408 TSGLCVWDPLMMSCASLL 1347 ILB409 LIMGCPWDPLLMLCQERR 1348 ILB410 SNPICIWDPLLMRCIWYQ 1349 ILB412 QSFLMRGCAWDPLLMQCI 1350 ILB414 LVVNCPWDPLLMACAVNV 1351 ILB418 STRDCYWDPLLMTCINYI 1352 ILB419 SYQVCCWDPLLMTCQRAR 1353 ILB421 YHWQLNGCVWHPLMINCL 1354 ILB423 LCLWDALLMECRIHSFDS 1355 ILB425 FCVWDPLLMIICRFTIIDRM 1356 ILB426 SMTMCGWNPLGMWCHSLG 1357 ILB428 TLVSLGLCAWDPLLMSCA 1358 ILB429 SFLFYSSCVWDPLLMQCG 1359 ILB430 TLDNCAWDPLLMRCWGQW 1360 ILB431 SSIYCLWDPLLMRCATPL 1361 ILB432 RCPWDPLMMMCIEEMYTD 1362 ILB436 LCSWDPLMMRCVVMDSLD 1363 ILB437 FFCLWHFCTGDPLKIHCM 1364 ILB439 QLFLTLNCSWDPLLMQCL 1365 ILB440 SYLGGLACAWDPLLMQCI 1366 ILB441 GFPECFWDPLLMDCTYWF 1367 ILB445 VFPWCYWDPLMMDCAIQN 1368 ILB450 FCVWDPLLMDCSGNTFYF 1369 ILB451 TCAWDPLLMVCRYPCPAF 1370 ILB452 AALECPWDPLMMSCIKTS 1371 ILB453 YLPSWLSCSWDPLLMICS 1372 ILB454 HLSLLMDCCWDPLTMQCT 1373 ILB455 RWPFCHWDPLLMMCSVSY 1374 ILB457 LPLLRFPCDGKQLWKFCL 1375 ILB461 QVSYCPWDPLLMQCQLSG 1376 ILB463 RSIECFWDPLMMECMGYV 1377 ILB464 LGMTFYVCPWDPLLMLCI 1378 ILB465 YYYSWPMCYWDPHMLFCL 1379 ILB467 PCYWDPLLMRCMLQVDNW 1380 ILB469 FFSDCNWDPLLMHCANKL 1381 ILB470 RPSLVPICYWDPLLMICI 1382 ILB471 NYFNCVWDPLRMDCQYFM 1383 ILB473 DWQYYSSKTYGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1384 ILB474 PDWITWQPAWGQAGQGSFNED CMWDPLLMDCAYSP 1385 ILB475 NMYHEHAAWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1386 ILB476 EVNHVEDFPAGQAGQGSFNED CMWDPLLMDCAYSP 1387 ILB477 WYTHSLQMWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1388 ILB478 GPDVTQYVKFGQAGQGSFNED CMWDPLLMDCAYSP 1389 ILB479 QSPSASHIMGGQAGQGSFNEDC MWDPLLMDCAYSP 1390 ILB480 GPVEESAPQYGQAGQGSATGGS SFNEDCMWDPLLMDCAYSP 1391 ILB481 WIMSLSWPMAGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1392 ILB482 HVGWHAMNPWGQAGQGSFNE DCMWDPLLMDCAYSP 1393 ILB483 VWTAFIQMNSGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1394 ILB485 PDWITWQPAWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1395 ILB486 VMWQWEEGHWGQAGQGSFNE DCMWDPLLMDCAYSP 1396 ILB487 TQEMWFTGINGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1397 ILB488 DNWWRIWDKQGQAGQGSFNE DCMWDPLLMDCAYSP 1398 ILB489 VTWFMYQDRAGQAGQGSFNE DCMWDPLLMDCAYSP 1399 ILB490 LPPPCYWTTWGKECQMFD 1400 ILB491 VEPIPYTMMRGQAGQGSFNEDC MWDPLLMDCAYSP 1401 ILB492 RIVVMEQMQIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1402 ILB493 MCNPMESECHGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1403 ILB494 VYWYDVKWTWGQAGQGSFNE DCMWDPLLMDCAYSP 1404 ILB495 DCEQNQTYCVGQAGQGSFNED CMWDPLLMDCAYSP 1405 ILB497 WWPNYTWDHPQGSATGGSSFN EDCMWDPLLMDCAYSP 1406 ILB498 DGSPGYRAYIISQFGQAGQGSA TGGSGSTASSGSGSSFNEDCMW DPLLMDCAYSP 1407 ILB499 WPWFEQEHFWGQAGQGSFNED CMWDPLLMDCAYSP 1408 ILB500 WCRPTDLKLVKGQAGQGSFNE DCMWDPLLMDCAYSP 1409 ILB501 DKSGGLVAIQGQAGQGSFNEDC MWDPLLMDCAYSP 1410 ILB502 DRWGWQKEPSGQAGQGSFNED CMWDPLLMDCAYSP 1411 ILB503 LAWKQSWIDTGQAGQGSATGG SGSTASSGSGSSFNEDCMWDPL LMDCAYSP 1412 ILB504 VMLEQMPMMRGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1413 ILB505 ATRMWPMSISGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1414 ILB506 MYVMKWQNWTGQAGQGSFNE DCMWDPLLMDCAYSP 1415 ILB508 ISDVYQWHSYGQAGQGSFNED CMWDPLLMDCAYSP 1416 ILB509 MVKLWEIYSWGQAGQGSFNED CMWDPLLMDCAYSP 1417 ILB511 QWGWSMWQEVGQAGQGSFNE DCMWDPLLMDCAYSP 1418 ILB512 MWSAVFQPYQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP GGSFNEDCMWDPLLMDCAYSP 1419 ILB513 AMHNVMWRAMGQAGQGSFNE DCMWDPLLMDCAYSP 1420 ILB514 HEDKFIVWMWGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1421 ILB516 QWWWTNSPWLGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1422 ILB517 QHQTAFTEMVGQAGQGSFNED CMWDPLLMDCAYSP 1423 ILB518 QTMWTRWMHDGQAGQGSFNE DCMWDPLLMDCAYSP 1424 ILB519 YAWHHHVLNTGQAGQGSFNED CMWDPLLMDCAYSP 1425 ILB520 YCPFMPALCVGQAGQGSATGG SGSTASSGSGSSFNEDCMWDPL LMDCAYSP 1426 ILB522 RTYSTPYWGWGQAGQGSFNED CMWDPLLMDCAYSP 1427 ILB523 EIFWWLLQWEGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1428 ILB524 QIDFKYMMWTGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1429 ILB525 QMQWLDDEFYGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1430 ILB526 PVQQEWMQWYGQAGQGSFNE DCMWDPLLMDCAYSP 1431 ILB527 QAWKLMWFAWGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1432 ILB528 MPSTQINQWVGQAGQGSFNED CMWDPLLMDCAYSP 1433 ILB529 TEKDPAQDWWGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1434 ILB531 EYTNTSWKGIIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1435 ILB532 QMEVAHVVDLGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1436 ILB533 IKPSSHWWWQGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1437 ILB534 YEEQQVDEFGGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1438 ILB535 AWWQGQRPYNGQAGQGSFNE DCMWDPLLMDCAYSP 1439 ILB536 PPLWMSQMVPQGSATGGSSFN EDCMWDPLLMDCAYSP 1440 ILB537 VEHLSHSYVLGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1441 ILB538 DCSPDVHDCHGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1442 ILB539 TYLQLWYFIQGQAGQGSFNEDC MWDPLLMDCAYSP 1443 ILB540 GRWWMVQWVEGQAGQGSFNE DCMWDPLLMDCAYSP 1444 ILB541 VMEGHQFAQFGQAGQGSFNED CMWDPLLMDCAYSP 1445 ILB542 WWSPSYAWVMGQAGQGSFNE DCMWDPLLMDCAYSP 1446 ILB543 MEMVSAAWQEGQAGQGSFNE DCMWDPLLMDCAYSP 1447 ILB544 YWWTIDYWPYGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1448 ILB545 YPMYYYQDISGQAGQGSFNED CMWDPLLMDCAYSP 1449 ILB546 IMWAGPIFPNGQAGQGSFNEDC MWDPLLMDCAYSP 1450 ILB547 IMGMYEGFRTGQAGQGSFNED CMWDPLLMDCAYSP 1451 ILB548 IERAYIISTRWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1452 ILB549 ETELKKPAMQGQAGQGSFNED CMWDPLLMDCAYSP 1453 ILB550 QDWHYPWVYQGQAGQGSFNE DCMWDPLLMDCAYSP 1454 ILB551 PLQKWEHIVNGQAGQGSFNED CMWDPLLMDCAYSP 1455 ILB552 TDSWCHTLYWALQHCVAQE 1456 ILB553 WQNPWPVRFWGQAGQGSATG GSGSTASSFNEDCMWDPLLMD CAYSP 1457 ILB554 APGVAMWMFIGQAGQGSFNED CMWDPLLMDCAYSP 1458 ILB555 QSNGAHAWVWGQAGQGSATG GSGSTASSFNEDCMWDPLLMD CAYSP 1459 ILB556 PDSSCHTLYWQLHHCARRE 1460 ILB557 KWNQQWTIARGQAGQGSFNED CMWDPLLMDCAYSP 1461 ILB558 ECSCAKSRYANGQAGQGSFNE DCMWDPLLMDCAYSP 1462 ILB559 SCQFLEEYKDCRGQAGQGSAT GGSSFNEDCMWDPLLMDCAYSP 1463 ILB560 VPSLIYHTYYGQAGQGSATGGS SFNEDCMWDPLLMDCAYSP 1464 ILB561 SIYISYRQNHGQAGQGSATGGS GSTASSFNEDCMWDPLLMDCA YSP 1465 ILB562 TYYTHTAWDRGQAGQGSFNED CMWDPLLMDCAYSP 1466 ILB563 LLVEFFKIITYGQAGQGSFNEDC MWDPLLMDCAYSP 1467 ILB564 FQGHWHIKKQGQAGQGSATGG SGSTASSGSGSSFNEDCMWDPL LMDCAYSP 1468 ILB565 FHLPIHWHYQGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1469 ILB566 LIVLQPKNMVGQAGQGSFNED CMWDPLLMDCAYSP 1470 ILB567 TYSWRDWGMYGQAGQGSFNE DCMWDPLLMDCAYSP 1471 ILB568 DGSHPIVVVTEWFGQAGQGSFN EDCMWDPLLMDCAYSP 1472 ILB569 VYWQAMNQDYGQAGQGSFNE DCMWDPLLMDCAYSP 1473 ILB570 PDSWCHTLYWDLLYCVNQE 1474 ILB572 DCAMWFMFKDQGQAGQGSAT GGSSFNEDCMWDPLLMDCAYSP 1475 ILB573 NNMHPLWPISGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1476 ILB574 FWYSWNPQWDGQAGQGSFNE DCMWDPLLMDCAYSP 1477 ILB576 EGYWRGPHWEGQAGQGSFNED CMWDPLLMDCAYSP 1478 ILB577 YRMKWDFMLHGQAGQGSFNE DCMWDPLLMDCAYSP 1479 ILB578 WWWDQHMLENGQAGQGSAT GGSGSTASSFNEDCMWDPLLM DCAYSP 1480 ILB579 PCHWAQQPCAGQAGQGSFNED CMWDPLLMDCAYSP 1481 ILB580 FFDFFSFSNSGQAGQGSATGGSS FNEDCMWDPLLMDCAYSP 1482 ILB581 VWTAFIQMNSGQAGQGSFNED CMWDPLLMDCAYSP 1483 ILB582 QIQQFSFWVQGQAGQGSFNEDC MWDPLLMDCAYSP 1484 ILB583 LFIPYMAQIWGQAGQGSFNEDC MWDPLLMDCAYSP 1485 ILB584 MTTMYHIIVLGQAGQGSATGGS GSTASSFNEDCMWDPLLMDCA YSP 1486 ILB585 GCSMCHMMMVKWHRGQAGQ GSFNEDCMWDPLLMDCAYSP 1487 ILB586 EQHVDWYWVPQGSFNEDCMW DPLLMDCAYSP 1488 ILB587 NQTQPKRPTDGQAGQGSFNED CMWDPLLMDCAYSP 1489 ILB588 ELGGRKSMLIGQAGQGSFNEDC MWDPLLMDCAYSP 1490 ILB589 EAWRELRWWNGQAGQGSFNE DCMWDPLLMDCAYSP 1491 ILB590 YCTDRVDICMGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1492 ILB591 LIVPAPLTWWGQAGQGSFNED CMWDPLLMDCAYSP 1493 ILB592 WVPDMWMHEWGQAGQGSFNE DCMWDPLLMDCAYSP 1494 ILB593 YQDSWITGMQGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1495 ILB594 YEEIICRLFPADWICSLLQ 1496 ILB595 VCQDEVYMWCEGQAGQGSAT GGSSFNEDCMWDPLLMDCAYSP 1497 ILB596 ADSWCHTLYWNLRHCEIQE 1498 ILB597 HHQQSMYWLEGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1499 ILB598 AEIITFALQYVGQAGQGSFNED CMWDPLLMDCAYSP 1500 ILB599 YTTYNWKPWQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1501 ILB600 ACDTQHHGCMGQAGQGSATG GSGSTASSFNEDCMWDPLLMD CAYSP 1502 ILB601 EVVGVGWFLNGQAGQGSFNED CMWDPLLMDCAYSP 1503 ILB602 PESWCHTLYWNLQHCLSQE 1504 ILB603 IDNKYEGMVLGQAGQGSFNED CMWDPLLMDCAYSP 1505 ILB604 HWMTYQFWIQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1506 ILB605 TCELMQAWTHLGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1507 ILB606 YYPQHIDHIMGQAGQGSATGGS SFNEDCMWDPLLMDCAYSP 1508 ILB607 MPLDDQPFLEGQAGQGSFNEDC MWDPLLMDCAYSP 1509 ILB608 HTEFQRLEWPQGSATGGSSFNE DCMWDPLLMDCAYSP 1510 ILB609 IQWPSMMEIHGQAGQGSFNEDC MWDPLLMDCAYSP 1511 ILB610 QTGWGPKYMWGQAGQGSFNE DCMWDPLLMDCAYSP 1512 ILB611 QSHIHLEKWLGQAGQGSFNEDC MWDPLLMDCAYSP 1513 ILB612 QCWIYPYDGLCNGQAGQGSAT GGSSFNEDCMWDPLLMDCAYSP 1514 ILB613 TCTHLVKTVLIGQAGQGSFNED CMWDPLLMDCAYSP 1515 ILB614 PLYTVWKWSGGQAGQGSATG GSGSTASSFNEDCMWDPLLMD CAYSP 1516 ILB615 FNFERWEQKPQGSFNEDCMWD PLLMDCAYSP 1517 ILB616 YVLQQVEMQEGQAGQGSFNED CMWDPLLMDCAYSP 1518 ILB617 SMTKYKALYHGQAGQGSFNED CMWDPLLMDCAYSP 1519 ILB618 QDNIAYQGWWGQAGQGSFNE DCMWDPLLMDCAYSP 1520 ILB619 MDHIWQHGQLGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1521 ILB620 SCFGDLQICWGQAGQGSATGGS GSTASSGSGSSFNEDCMWDPLL MDCAYSP 1522 ILB621 HCKDMHWAIQFGQAGQGSFNE DCMWDPLLMDCAYSP 1523 ILB622 WLFVAEQSQYGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1524 ILB623 ANSWCIITLYWDLMYCVWQE 1525 ILB624 DGSGFYPNKWFHGGQAGQGSA TGGSSFNEDCMWDPLLMDCAY SP 1526 ILB625 MVYRWVMQADGQAGQGSATG GSGSTASSGSGSSFNEDCMWDP LLMDCAYSP 1527 ILB627 HPMEIKTMMKGQAGQGSFNED CMWDPLLMDCAYSP 1528 ILB629 LQAWMWQVNQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1529 ILB630 GCNYFPTQWECYGQAGQGSFN EDCMWDPLLMDCAYSP 1530 ILB631 LQAWMWQVNQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP QGSATGGSSFNEDCMWDPLLM DCAYSP 1531 ILB632 QFPYESPPSIGQAGQGSFNEDC MWDPLLMDCAYSP 1532 ILB633 IYLSWDMPALGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1533 ILB634 QYTEMVALVYGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1534 ILB635 PDSWCHTLYWHLQHCLSQE 1535 ILB636 PAKDPFVMRHGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1536 ILB637 WQVSSVQLPYGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1537 ILB638 QQTQTYWGTWGQAGQGSFNE DCMWDPLLMDCAYSP 1538 ILB639 DQLWRQNWMQGQAGQGSFNE DCMWDPLLMDCAYSP 1539 ILB640 QYYNVYWQNQGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1540 ILB641 QTYSNKPMGPQGSFNEDCMWD PLLMDCAYSP 1541 ILB642 EEEWSWYDRRGQAGQGSFNED CMWDPLLMDCAYSP 1542 ILB643 AYYWKEWMKMGQAGQGSAT GGSSFNEDCMWDPLLMDCAYS PG 1543 ILB644 AIAHLDVFPPQGSFNEDCMWDP LLMDCAYSP 1544 ILB645 QYQSRQTFQIGQAGQGSFNEDC MWDPLLMDCAYSPQGSFNEDC MWDPLLMDCAYSP 1545 ILB646 YWAWQQEFQWGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP GSYWAWQQEFQWGQAGQGSA TGGSSFNEDCMWDPLLMDCAY SP 1546 ILB647 YYQGFPWFQIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1547 ILB648 FFYQWSYGWSGQAGQGSFNED CMWDPLLMDCAYSP 1548 ILB649 YEEIICALFPTDWICTLII 1549 ILB650 WRWHAEWDWYGQAGQGSAT GGSSFNEDCMWDPLLMDCAYSP 1550 ILB652 WRWEFGYYYHGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1551 ILB653 MFYEYQWWIHGQAGQGSFNED CMWDPLLMDCAYSP 1552 ILB654 PDWITWQPAWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPQ GSATGGSSFNEDCMWDPLLMD CAYSP 1553 ILB655 WPFYEYIIHMGQAGQGSATGGS SFNEDCMWDPLLMDCAYSP 1554 ILB656 RPYYYQWHIWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1555 ILB657 TGFFWSWSFGGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1556 ILB659 SKMEQYPWNMGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP QGSATGGSSFNEDCMWDPLLM DCAYSP 1557 ILB660 YHWQQQHQYHGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP QGSATGGSSFNEDCMWDPLLM DCAYSP 1558 ILB661 DWQVYYKTWIGQAGQGSATG GSGSTASSGSGSSFNEDCMWDP LLMDCAYSPQGSATGGSGSTAS SGSGSSFNEDCMWDPLLMDCA YSP 1559 ILB662 YRYTTYWWYSGQAGQGSATG GSGSTASSFNEDCMWDPLLMD CAYSP 1560 ILB664 QCCMSQVSWYVGQAGQGSAT GGSSFNEDCMWDPLLMDCAYS PQGSATGGSSFNEDCMWDPLL MDCAYSPQGSATGGSSFNEDC MWDPLLMDCAYSP 1561 ILB665 YYQGFPWFQIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPG GGSYYQGFPWFQIGQAGQGSA TGGSSFNEDCMWDPLLMDCAY SP 1562 ILB666 QWSFHWWSHTGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1563 ILB667 WTFEWWIYTKGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1564 ILB669 ADRFFVWNPYGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPQ GSATGGSSFNEDCMWDPLLMD CAYSP 1565 ILB670 QRYMMQTMLIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPQ GSATGGSSFNEDCMWDPLLMD CAYSP 1566 ILB671 PWSKATQPGWGQAGQGSATGG SGSTASSFNEDCMWDPLLMDC AYSPQGSATGGSGSTASSFNED CMWDPLLMDCAYSPQGSATGG SGSTASSFNEDCMWDPLLMDC AYSP 1567 ILB672 IWWGYSWDHYGQAGQGSATG GSSFNEDCMWDPLLMDCAYSP 1568 ILB673 IVNFTWNFLSGQAGQGSFNEDC MWDPLLMDCAYSP 1569 ILB675 MQFSWTQHFFGQAGQGSATGG SGSTASSGSGSSFNEDCMWDPL LMDCAYSPGMQFSWTQHFFGQ AGQGSATGGSGSTASSGSGSSF NEDCMWDPLLMDCAYSP 1570 ILB676 LHANSIATNWGQAGQGSATGG SSFNEDCMWDPLLMDCAYSP 1571 ILB677 PDSWCHTLYWQLIHCERQE 1572 ILB678 QFPYESPPSIGQAGQGSATGGSS FNEDCMWDPLLMDCAYSP 1573 ILB680 YYQGFPWFQIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPG GGSYYQGFPWFQTGQAGQGSA TGGSSFNEDCMWDPLLMDCAY SPGGGSYYQGFPWFQIGQAGQG SATGGSSFNEDCMWDPLLMDC AYSP 1574 ILB681 YYQGFPWFQIGQAGQGSATGG SSFNEDCMWDPLLMDCAYSPG GGSYYQGFPWFQIGQAGQGSA TGGSSFNEDCMWDPLLMDCAY SP 1575 ILB739 GEAWCWDPIRMDVRVCSVFI 1576 ILB740 GPYSCPWDPMKMECAYSP 1577 ILB741 EYSSCNWDPLLMQCQRVV 1578 ILB742 FHSTCFWDPLLMRCVSAP 1579 ILB743 SVSPCIWDPLLMMCGNYS 1580 ILB744 VGVDCPWDPLMMECAGRN 1581 ILB745 VSAECYWDPLMMQCSTDM 1582 ILB747 FGDTCFWHPFNEDMCESKP 1583 ILB748 QWLHCFWDPLMMKCVTVS 1584 ILB749 TPHPCHWDPMIMYCMLEM 1585 ILB750 HEEDCWWDPLIMQCFNRT 1586 ILB751 HTANCPWDPLIMNCSFQS 1587 ILB752 YTKHCQRLPTYKICNLFM 1588 ILB755 ADSWCHTLYWQLQHCLSQE 1589 ILB756 HSRWCYWEPIRMIEMCMGDK 1590 ILB757 PGVACFWDPLLMSCSYDE 1591 ILB758 RAFTCPWDPLIMDCAGAS 1592 ILB759 GDDWCAWDPIRMQCSYTK 1593 ILB760 EIEQCWWDPMLMEGVCWPSI 1594 ILB761 HGRSCAWDPLKMDCVWYH 1595 ILB762 RLGACVWDPLMMQCAWIS 1596 ILB763 LYPSCPWDPILMQCSALK 1597 ILB764 PTPLCHWDPLLMHCTASD 1598 ILB765 TDSWCHTLYWQLHHCAILE 1599 ILB766 RWNVCAWDPLLMQCASAP 1600 ILB767 MSVSCNWDPLLMTCRLPT 1601 ILB768 PGLRCPWDPLMMTCVAHP 1602 ILB769 QIAFCLWDPLLMDCTVQT 1603 ILB770 SPQECPWDPIAMKCAFVR 1604 ILB771 TSHPCWWDPIMMSCWLEE 1605 ILB772 VQLYCIWDPLMMDCGQRS 1606 ILB773 CRNSCHWDPLIMDCMQEM 1607 ILB774 FNEDCMWDPLLMDCAYSPGQG SAAGGGSIVQHQPSRMY 1608 ILB775 FNEDCMWDPLLMDCAYSPAGG GSICQATPYSPWCR 1609 ILB776 HRAPCFSCTACRQPCFSLT 1610 ILB778 FNEDCMWDPLLMDCAYSPAGG SALRMCDSWVENRCLG 1611 ILB779 FNEDCMWDPLLMDCAYSPGQG SAAGGSAQWQCHFNPTGMACV DMLQG 1612 ILB780 FNEDCMWDPLLMDCAYSPGQG SAAGGGSIIPWRIINMQKI 1613 ILB781 FNEDCMWDPLLMDCAYSPGQG SAAGGGSQFPYESPPSI 1614 ILB782 FNEDCMWDPLLMDCAYSPAGG GSVCPTQPANCQ 1615 ILB783 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPCRFWPGRAFCH 1616 ILB784 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSECELDYHMV WCK 1617 ILB785 FNEDCMWDPLLMDCAYSPGQG SAAGGGSFWMQQAQQHK 1618 ILB786 FNEDCMWDPLLMDCAYSPGQG SAAGGGSKCDWQPWFCP 1619 ILB787 FNEDCMWDPLLMDCAYSPAGG GSFVGESQYMQTK 1620 ILB788 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSQPWR HNMQKI 1621 ILB789 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSQFPYE SPPSI 1622 ILB791 FNEDCMWDPLLMDCAYSPGRG SSLGGVSPPTVSGRASA 1623 ILB792 ANSWCHTLYWQLHHCEILE 1624 ILB793 FNEDCMWDPLLMDCAYSPAGG GSIWQTEVIVPD 1625 ILB794 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSRVRLD HWEQV 1626 ILB797 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGDGSIEHHGAWSAQ 1627 ILB798 FNEDCMWDPLLMDCAYSPGQG SAAGGSPVLGCRNVHNTQCRL WFQG 1628 ILB799 GLHGCQYVQMFFHFCAPKT 1629 ILB800 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSHWERCVPHSD PDYNCSFDS 1630 ILB801 FNEDCMWDPLLMDCAYSPAGG SGWVFRCKNMPCAKAEH 1631 ILB802 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPPHPSNSIWR 1632 ILB803 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSRLSLHCPEHPC GSSWTQG 1633 ILB804 FNEDCMWDPLLMDCAYSPAGG GSPDWITWQPAW 1634 ILB806 FQGHCALFPTDWICTLKS 1635 ILB807 FNEDCMWDPLLMDCAYSPGQG SAAGGSVGLLCQDFAYGQTHC DVLQQG 1636 ILB808 FNEDCMWDPLLMDCAYSPGQG SAAGDGSMCYQEPHFTYCP 1637 ILB809 FNEDCMWDPLLMDCAYSPGQG SAAGGGSECMSSWHGCT 1638 ILB810 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGSDAWGC LDDFFMKVAGCKTLF 1639 ILB811 FNEDCMWDPLLMDCAYSPGQG SAAGGSQFLLCNGSEWCPANR QG 1640 ILB812 FNEDCMWDPLLMDCAYSPGQG SAAGGGSFCMWPFCIPPR 1641 ILB813 FNEDCMWDPLLMDCAYSPGQG SAAGGGSMGGEDDIWHQ 1642 ILB814 PDSRCHTLYWALRYCAYQE 1643 ILB815 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSWHSYTAMYAD 1644 ILB816 FNEDCMWDPLLMDCAYSPGQG SAAGGGSGMVSHWKKVQ 1645 ILB817 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGSQGMPC QYHAMQLCPPSR 1646 ILB818 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSHQHNF QLRYT 1647 ILB819 FNEDCMWDPLLMDCAYSPGQG SAAGGGSLCDQWQSARYCV 1648 ILB820 FNEDCMWDPLLMDCAYSP 1649 ILB821 FNEDCMWDPLLMDCAYSPGQG SAAGGGSYCHEDPRSLDCR 1650 ILB822 FNEDCMWDPLLMDCAYSPGQG SAAGGGSHPWRHNMHKI 1651 ILB823 FNEDCMWDPLLMDCAYSPGQG SAAGGSPLVDCLTYPEWVNCPR TIQG 1652 ILB824 FNEDCMWDPLLMDCAYSPGQG SAAGGGSDCNFAPKETNCW 1653 ILB825 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGSKTLFCG YDGYDMCDLSA 1654 ILB826 FNEDCMWDPLLMDCAYSPGQG SAAGGGSWNRHPPPSGQ 1655 ILB827 FNEDCMWDPLLMDCAYSPAGG GSMQYHPWQDMW 1656 ILB828 FNEDCMWDPLLMDCAYSPGQG SAAGGGSSCNITDQMGVCL 1657 ILB829 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSWDDTCNAEVY QQGLMCDTRVQG 1658 ILB831 FNEDCMWDPLLMDCAYSPGQG SAAGGSARRGCVMLNECSSVS QG 1659 ILB832 FNEDCMWDPLLMDCAYSPGQG SAAGGSRMRSVCPHHVCWRGV PQG 1660 ILB833 FNEDCMWDPLLMDCAYSPAGG SPKSECVAPLEMIECPSLW 1661 ILB834 FNEDCMWDPLLMDCAYSPGQG SAAGGSSVFWCYSQHGCALTS QG 1662 ILB835 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPRAWQFPVSS 1663 ILB836 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSICMEYPIIRPCT 1664 ILB837 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPCLSWYVAPMF 1665 ILB838 FNEDCMWDPLLMDCAYSPGQG SAAGGSADDCCVSHLPFIGYCG FVRQG 1666 ILB840 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGSGEDQCK LFGSGLDTCIPYV 1667 ILB842 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPCVKAPEFDWCT 1668 ILB843 FNEDCMWDPLLMDCAYSPGQG SAAGGSHESCAVHYQHGCPYA SQG 1669 ILB844 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGNCSCFIKPV GPDCWAVS 1670 ILB845 QAASAPASEPPGSLCLPDQFRC GNGQCIPLDWVCDGVNDCPDD SDEEGCPPRTCAPSQFQCGSGY CISQRWVCDGENDCEDGSDEA NCAGSVPTCPSDEFR 1671 ILB846 FNEDCMWDPLLMDCAYSPGQG SAAGGGSEEQPTEWHVY 1672 ILB847 FNEDCMWDPLLMDCAYSPAGG SETFDCNTPWEHECYGRF 1673 ILB848 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSETLPCQYWGQ SWCSQSAQG 1674 ILB849 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSPCTPW TEWQQCP 1675 ILB850 FNEDCMWDPLLMDCAYSPGQG SAAGGSHSMLCQPWHLCPPAQ QG 1676 ILB851 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSKCSID VQQCM 1677 ILB852 FNEDCMWDPLLMDCAYSPGQG SAAGGGSQPWRHNMQKI 1678 ILB853 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSHHKNNGTIYQ 1679 ILB854 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPAERMKMHMF 1680 ILB855 FNEDCMWDPLLMDCAYSPGQG SAAGGGSREFTQNWSIY 1681 ILB856 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSAYTM WQYHKF 1682 ILB857 FNEDCMWDPLLMDCAYSPGQG SAAGGGSYMGQIPAHNI 1683 ILB858 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSKCWSFSGNRP CP 1684 ILB860 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPDWITWQPAW 1685 ILB861 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSYQPGCAQWW DMERGCFITQ 1686 ILB862 FNEDCMWDPLLMDCAYSPGQG SAAGGGSSYWEHGWPWQ 1687 ILB863 FNEDCMWDPLLMDCAYSPGQG SAAGGGSWPSPPWKQMV 1688 ILB864 FNEDCMWDPLLMDCAYSPAGG GSDEFQFGSEMM 1689 ILB865 FNEDCMWDPLLMDCAYSPGQG SAAGGGSINALGVAVQH 1690 ILB866 FNEDCMWDPLLMDCAYSPGQG SAAGGGSQWQWKIHPAE 1691 ILB867 FNEDCMWDPLLMDCAYSPGQG SAAGGGSLCERPLHTPWA 1692 ILB868 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPWAKNRHEAV 1693 ILB869 FNEDCMWDPLLMDCAYSPAGG GSQPWRHNMQKI 1694 ILB870 FNEDCMWDPLLMDCAYSPAGG SIRGLSCWPQDCGLEEL 1695 ILB871 FNEDCMWDPLLMDCAYSPGQG SAAGGGSLNFGSYSTPH 1696 ILB872 FNEDCMWDPLLMDCAYSPGQG SAAGGGSHYLWQEKTYK 1697 ILB873 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSDCGA NHEHCS 1698 ILB874 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSRVQIIV PEKW 1699 ILB875 FNEDCMWDPLLMDCAYSPGQG SAAGGGSEHHWEHPTNF 1700 ILB876 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSSAIIDCTEESWC WMEVQG 1701 ILB877 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSYSFRCDVEPHQ PGCFDLIQG 1702 ILB878 FNEDCMWDPLLMDCAYSPGQG SAAGGGSLMYMWEPRTQ 1703 ILB879 FNEDCMWDPLLMDCAYSPGQG SAAGGSLEASCAQSDQCFRQSQG 1704 ILB880 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSLCFSN WDEMKCF 1705 ILB881 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSICNWEEVDPH CA 1706 ILB882 FNEDCMWDPLLMDCAYSPGQG SAAGGGSPWLDPQWDIW 1707 ILB883 FNEDCMWDPLLMDCAYSPAGG SRNSWICMEWHCSDLTR 1708 ILB884 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSWGYQQSWV QQ 1709 ILB885 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSLTVSNCQEEIC MQDEYQG 1710 ILB886 FNEDCMWDPLLMDCAYSPGQG SAAGGGSQCKAYMPQCA 1711 ILB887 FNEDCMWDPLLMDCAYSPAGG GSSWQRHDGHQI 1712 ILB888 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSTREMLVHPRH 1713 ILB889 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSQQKER ISPWP 1714 ILB890 FNEDCMWDPLLMDCAYSPGQG SAAGGGSFNINNIYVGN 1715 ILB891 FNEDCMWDPLLMDCAYSPGQG SAAGGGSVSSYNPWIIY 1716 ILB892 FNEDCMWDPLLMDCAYSPGQG SAAGGSMQTSCEFHEKYERHG CQQLSQG 1717 ILB893 FNEDCMWDPLLMDCAYSPGQG SAAGGGSNCLIAEWECE 1718 ILB894 FNEDCMWDPLLMDCAYSPGQG SAAGGGSWEVPRSLWGQGQAG PGGGSG 1719 ILB895 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGSMSLECE SYGIGWCSLLII 1720 ILB896 FNEDCMWDPLLMDCAYSPGQG SAAGGSHISTCKLIMGAYAHCQ TPSQG 1721 ILB897 FNEDCMWDPLLMDCAYSPGQG SAAGGGSLQQGRRHHMR 1722 ILB898 FNEDCMWDPLLMDCAYSPGQG SAAGGSNIDFQCYPFKCGVLFS QG 1723 ILB899 FNEDCMWDPLLMDCAYSPGQG SAAGGGSGCSSTPFAWSCS 1724 ILB900 FNEDCMWDPLLMDCAYSPGQG SATGGSGSTASAAGGGSPCQNQ PSYQVCT 1725 ILB901 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSLRQSCYPIPESE KCSFSNQG 1726 ILB902 FNEDCMWDPLLMDCAYSPGQG SAAGGGSFFHWPTKAKE 1727 ILB903 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSQCEFEQEECW 1728 ILB904 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGSGVTGCSGFPTT GTCSQHEQG 1729 ILB905 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSLDYHDIPRHA 1730 ILB906 FNEDCMWDPLLMDCAYSPGQG SATGGSAAGGGSSGWEHSSIVF 1731 ILB907 FNEDCMWDPLLMDCAYSPGQG SAAGGGSWWSPAYGWQN 1732 ILB908 FNEDCMWDPLLMDCAYSPGQG SAAGGGSQCALKSFTVPCN

TABLE 14 IL6R binding MRDs SEQ ID: Ref. No. Peptide Sequence 1747 ILC19 GDWEWVWDWCF 1748 ILC20 PCQWVIYHCF 1749 ILC21 WEIEWSWYWD 1750 ILC23 FYMEFIIWSP 1751 ILC24 WYWMLEWEWD 1752 ILC25 PCEEAFAAYLE 1753 ILC27 QCFAWEFVWHCD 1754 ILC28 YTWDLYIYQA 1755 ILC31 MYTEWQMWWIGQAGQGGGSG GGGSFYDQWEVELH 1756 ILC33 AEFDWMQYGYGQAGQGGGSG GGGSYEFNWKYQEW 1757 ILC34 YMQWKVEWWG 1758 ILC35 WEITWDWEYY 1759 ILC39 WCLITPWEVQCY 1760 ILC40 TTMVFQASLG 1761 ILC41 LCFCYEYEWVH 1762 ILC42 HWEFYYEFGI 1763 ILC43 LCYWEVWFTWE 1764 ILC44 PCYLVMMEWECM 1765 ILC46 GCDFYMNLCK 1766 ILC50 WQEQWWYKQH 1767 ILC52 SELSCYWEMKWECSWVT 1768 ILC53 FCWSCEVFQMEICDELG 1769 ILC55 HALHCANEERIFVQCLESS 1770 ILC56 VYLECNVYEWPEAWCVVMP 1771 ILC57 TQHLCAWDVRSQADMCIYWT 1772 ILC58 QVYVCYVLAEQHEAICTSWA 1773 ILC59 LTWVCTVMQHMNHMECLLDL 1774 ILC60 MLLHCPAMWKCQSDL 1775 ILC61 LTWDCYQVFEEWYDCTMSII 1776 ILC62 VWGGCWYHFTIQHWCPTLS 1777 ILC63 MTILCYEQREDMWDCWNVF 1778 ILC64 LDCACHFEEHVRTIFCSIGH 1779 ILC65 WLVDCWVVEDMNMICRLYE 1780 ILC66 LDFLCYENIHNNFDCFRVM 1781 ILC67 VASHCIQMPHWLLCQTLI 1782 ILC70 WTVWCEWENEYGGWCDVSL 1783 ILC71 LQCKCDEPYPSYYTCRVYF 1784 ILC74 GPFMCMMDLHRDELLHCWASD 1785 ILC75 VAVDCFWVTPYEPWYCTVMS 1786 ILC77 MTDRCYTYEWQYTVCYGRN 1787 ILC80 RDWRCYLMAHWVHVCGPFIGQ AGQGGPGWAGGSDMDLCQDW VYESKYCVPLA 1788 ILC83 SRYVICDLDMVFRECHGAF 1789 ILC84 SALVLCELEMVFRECHGVL 1790 ILC86 SEFILCDLAMVFAECHGAA 1791 ILC89 SELLLCAFDMVISECHGTT 1792 ILC90 DSFGCTWEVWGRECHPQL 1793 ILC93 SEFIFCDLEMVFRECIIGTT 1794 ILC95 SEFVYCMLDMVFRECHGTA 1795 ILC96 SELILCALEMVFNECHGAV 1796 ILC97 SDLVLCEWDMVVRECHGVF 1797 ILC99 SDLIWCELDMVFRECHGAA 1798 ILC102 SSFVFCDLEMVFRECHGVF 1799 ILC103 SDLILCNLEMVFKECHGVI 1800 ILC106 SSLILCDLEMVFKECHGVL 1801 ILC108 SEIVLCEWAMVLAECHGAA 1802 ILC144 SRCDRVLGTHINL 1803 ILC164 SHEEWTCFPYLCTFWAE 1804 ILC165 SEEEWTCFPYLCSIWLE 1805 ILC166 SQEEWTCFPYLCSYWLE 1806 ILC167 SQEEWTCFPYLCSFWSE 1807 ILC169 SHEEWTCFPYLCKYWLH 1808 ILC170 SHEDWTCFPYLCKVLLH 1809 ILC171 SHEEWTCFPYLCENWLH 1810 ILC172 SEEEWTCFPYLCTDWVH 1811 ILC173 SEEEWTCFPYLCTILLQ 1812 ILC174 SDEEWTCFPYLCSVWVE 1813 ILC175 SEEEWTCFPYLCSYLLH 1814 ILC176 SHEDWTCFPYLCKYLLD 1815 ILC177 SQEEWTCFPYLCREWVE 1816 ILC178 SDEEWTCFPYLCSQWVE 1817 ILC179 SEEEWTCFPYLCREWAH 1818 ILC180 SEEEWTCFPYLCSYWLD 1819 ILC181 SEEEWTCFPYLCSQWLE 1820 ILC182 SEEEWTCFPYLCTNWLH 1821 ILC183 SHEDWTCPPYLCRILVE 1822 ILC184 SDEEWTCFPSLCTQLLQ 1823 ILC185 SEEDWTCFPYLCRILVE 1824 ILC186 SHEEWTCFPYLCGKWAE 1825 ILC187 SIIEDWTCFPYLCSILLQ 1826 ILC189 SHEDWTCPPYLCRVLLE 1827 ILC190 SDEEWTCFPYLCTFWLQ 1828 ILC191 SEEEWTCFPYLCRNWLD 1829 ILC192 SEEEWTCFPYLCGLWVE 1830 ILC193 SEEDWTCFPYLCRILLD 1831 ILC194 SHEDWTCFPYLCRILSH 1832 ILC195 SEEEWTCFPYLCTNWLD 1833 ILC196 SEEEWTCFPYLCSDWLE 1834 ILC197 SEEEWTCFPYLCTHWLH 1835 ILC198 SEEEWTCFPSLCNFWSQ 1836 ILC199 SQEEWTCFPYLCTYWLQ 1837 ILC200 SHEEWTCFPYLCTYWSH 1838 ILC201 SDEEWTCFPYLCSIWAQ 1839 ILC202 SDEEWTCFPYLCSYWVD 1840 ILC203 SHEEWTCFPYLCRILLD 1841 ILC204 SQEEWTCFPYLCTDWLH 1842 ILC205 SEEEWTCFPYLCSFWLE 1843 ILC206 SHEDWTCFPYLCTLLLQ 1844 ILC207 SHEDWTCFPYLCNILLQ 1845 ILC208 SEEEWTCFPYLCSHWAE 1846 ILC209 SHEEWTCFPYLCKILLE 1847 ILC210 SQEEWTCFPYLCSYWLQ 1848 ILC211 SEEEWTCFPYLCSIWLD 1849 ILC212 SHEEWTCFPTLCTDWLQ 1850 ILC213 SEEEWTCFPSLCTIWLQ 1851 ILC214 SHEEWTCFPYLCSDWLQ 1852 ILC215 SEEEWTCFPYLCSLWVD 1853 ILC216 SHEEWTCFPYLCSVWAQ 1854 ILC218 SHEEWTCFPYLCSLWAQ 1855 ILC219 SDEEWTCFPTLCTDWLQ 1856 ILC220 SQEEWTCFPYLCSHWLD 1857 ILC221 SEAEWTCFPYLCSDWLH 1858 ILC222 SEEEWTCFPYLCGQWVE 1859 ILC223 SDEEWTCFPSLCSFWLE 1860 ILC224 SEEEWTCFPYLCTLWSQ 1861 ILC225 SDEEWTCFPTLCSLWSE 1862 ILC226 SDEEWTCFPSLCSFWLD 1863 ILC227 SQEEWTCFPYLCTDWVQ 1864 ILC228 SEEEWTCFPYLCSILVD 1865 ILC229 SEEEWTCFPYLCSKLLH 1866 ILC230 SEEEWTCFPYLCGKWLQ 1867 ILC231 SEEEWTCFPYLCTQLLH 1868 ILC232 SDEDWTCFPYLCRILLE 1869 ILC233 SQEEWTCFPSLCSDWVQ 1870 ILC234 SDEEWTCFPYLCSFWAQ 1871 ILC236 SEEEWTCFPYLCRELLE 1872 ILC237 SEEEWTCFPSLCGVWAE 1873 ILC238 SHEEWTCFPYLCTVWAE 1874 ILC239 SEEEWTCFPYLCTNLVE 1875 ILC240 SQEEWTCFPYLCSKWVD 1876 ILC241 SQEEWTCFPYLCSDWLQ 1877 ILC242 SDEEWTCFPYLCSYWLQ 1878 ILC243 SDEEWTCVPSLCAKLADGQAG QAAAGGSHEEWTCFAYLCGLL AD 1879 ILC244 SHEEWTCFPYLCTYWLQ 1880 ILC247 SEEEWTCFPYLCSEWAQ 1881 ILC248 SEEEWTCFPYLCSQWLH 1882 ILC249 SDEEWTCFPYLCSEWLQ 1883 ILC250 SACEEAFAAYLEDSAV 1884 ILC253 SDEEWTCFPYLCEYWLQ 1885 ILC254 SQEEWTCFPYLCRILVD 1886 ILC257 SHEEWTCFPYLCSYLLE 1887 ILC258 SEEEWTCFPSLCKILLQ 1888 ILC259 SEEEWTCFPYLCSKWVE 1889 ILC260 SACEEAFAAYLEDGLM 1890 ILC262 SEEEWTCFPSLCSNLVQ 1891 ILC263 SHEEWTCFPYLCSFWAE 1892 ILC264 SHEEWTCFPSLCTLWSH 1893 ILC265 SEEEWTCFPYLCSKWLD 1894 ILC269 SEEEWTCFPYLCTYWVD 1895 ILC270 SDEEWTCFPYLCDYWLII 1896 ILC274 SHEEWTCFPYLCSFWLE 1897 ILC275 SDEEWTCFPYLCTDWLH 1898 ILC276 SHEEWTCFPYLCNLLSH 1899 ILC277 SQDEWTCFPSLCSDLSE 1900 ILC280 SEEEWTCFPYLCSFWLQ 1901 ILC281 SEEEWTCFPYLCSLWLE 1902 ILC282 SHEEWTCFPYLCSYWLE 1903 ILC283 FMV HCTWYDVTID CVGPT 1904 ILC284 SDI SCSYIDGWYQ CLLHL 1905 ILC285 WGC LCQQQGHFME CEITW 1906 ILC286 ESL LCIQHDVWVD CYIVE 1907 ILC287 NSS FCYIHAIEWV CESSS 1908 ILC288 DLL YCEWEMVVRE CHGTIGQ 1909 ILC292 QIVECWTEMDWHHCVLFF

TABLE 15 IL17a binding MRDs SEQ ID: Ref. No. Peptide Sequence 1932 ILA1 LSLWCWHDQAEFCVESN 1933 ILA2 SKYDCFWEHEWLYCAPRM 1934 ILA3 FDLYCWVWPDMMECTAGT 1935 ILA4 GTWDCWVEGEWVYCTPWS 1936 ILA5 STEGCHQDWYDLCAFLA 1937 ILA6 LSLWCWMFPEHNACLSPV 1938 ILA7 ESLWCWMFPNEQTCLVQH 1939 ILA8 VSYLCDEYQGLLYCMVQR 1940 ILA9 LDLWCWFPNDTKCMRLT 1941 ILA10 LGLWCFVYPDDQMCIRSW 1942 ILA11 MGELCEFFGFHDCPMQI 1943 ILA12 SETLCWDGWDIWICQDPQ 1944 ILA13 DWATCWEFNELWYCIPYN 1945 ILA16 IDPGCWLTPWSFQCPGYM 1946 ILA19 LGLWCWMFPIDSFCYPAV 1947 ILA23 LHLWCWMFPLDDACADFF 1948 ILA24 LDLWCWFPNDDWCMTHL 1949 ILA25 MVLDCYIIEELDEYWCDWYS 1950 ILA26 LGLWCWMFPIDSFCYPAI 1951 ILA27 YDVWCFFFPDEIMCNETQ 1952 ILA28 VEIDCEYDWMNEYLYCAPRV 1953 ILA29 KCNFNPWKAACG 1954 ILA30 DWLHCEIKPPFYHCWNMT 1955 ILA31 LSLWCWINDVNCSEPV 1956 ILA33 LSLWCFIDETSEQCSILS 1957 ILA34 MDLSCWMTDPTLEWWFCEYRP 1958 ILA35 LNLWCWMFPAAAECEFDQ 1959 ILA36 WCFGCVIPPFWYECMCPT 1960 ILA37 LTLWCWIFPQDEICWTML 1961 ILA38 IFFDCTFSDWQLDCVELD 1962 ILA39 LALWCWMFPMSDECPPKH 1963 ILA40 GHIGCWGPEWYCHWYG 1964 ILA43 VDELCVFMFGQSDCRNLF 1965 ILA44 LSLWCWAFPSDTVCLARL 1966 ILA45 STWECWEVNNETYECARLL 1967 ILA46 SNPWCHIAEYELWCWMGH 1968 ILA47 TSWECHETGDDYFYCWHLK 1969 ILA48 LDLVCWMYPTEKACWSMR 1970 ILA50 LGLWCWMFPWEQVCSVRL 1971 ILA51 TWYLCWESQWWDVYYCEEEG 1972 ILA52 MSLWCWMFPWDNMCFKFS 1973 ILA53 IRDDCQENWQSELVCFLLG 1974 ILA55 LGLWCWMFPMDPDCHRGY 1975 ILA56 LAYECWVLEDLEYVICEQGM 1976 ILA57 LFFICYETPEEYMYCLPKG 1977 ILA58 QSPWCFMFPQEEHCNHYN 1978 ILA59 LSLWCWIFPTEETCKHGL 1979 ILA61 HSAWCWFPEDNICLDRR 1980 ILA62 LTMWCEEMIKDEWWCVPAI 1981 ILA63 NALWCWFPEASECGLME 1982 ILA64 YTYYCVETEYNMVCELDY 1983 ILA65 IGWQCEYLPWDECQWQF 1984 ILA66 DDLFCWMFPSAYVCLPPV 1985 ILA67 QDELCNFIFNDHCASAF 1986 ILA68 LQELCHYLGFEECVGQQ 1987 ILA69 ASLWCWMYPNDISCFMES 1988 ILA71 TADLCMFLWHQDCGRIF 1989 ILA73 LSLWCWLHDQAEFCVESN 1990 ILA74 WPLECYTDEE LYDIWCLMDL

TABLE 16 TL1A binding MRDs SEQ ID: Ref. No. Peptide Sequence 1993 TLA26 LLGDCFAGFDNIWCEFLD 1994 TLA29 KCNFNPWKAACG 1995 TLA30 QTFWCYDDWHECSGKP 1996 TLA34 LNHSCWWWDEWEECPHNV 1997 TLA37 KVEYCYDDWHQCKQVQ 1998 TLA38 DDSMCAWWDEWHECEHHS 1999 TLA39 EYMYCYDDWYDC 2000 TLA43 DNTDCYYQGYWVYCTDLN 2001 TLA44 RSDVCWDMAAQHLFYCGEWI 2002 TLA46 RYDVCWDQKLQELYYCSYYH 2003 TLA47 LSSVCWNNSAEKLEYCGPLE 2004 TLA49 HITICDYQEYFYMCTILY 2005 TLA50 DMAGCHWDPVYINFDCRASM 2006 TLA56 KFNECFVGYDYIWCNSSV 2007 TLA58 GEDTCHWVHFIDDWYCLAPM 2008 TLA62 EVTICQYTEDFTYYYCTRPN 2009 TLA70 EVCWDSESESIQWCDMEW 2010 TLA72 PFVVCYTQWQGEREFHNCWKLQ 2011 TLA77 QTVRVCFYDRCQDVKF 2012 TLA81 QKAICWNTEHITYYYCSRPN 2013 TLA85 SSWDCMGGPIVWVCPEGR 2014 TLA88 RVEECSYEFRQCLANY 2015 TLA89 DYDVCWDVLADRLYYCDEPI 2016 TLA90 DSFGCTWEVWGRECHPQL 2017 TLA91 DYDVCWDLEVDRLYYCDDPL 2018 TLA92 DYDVCWDVQLDRLYYCDVPM 2019 TLA93 DYDVCWDIHADRLYYCNAPF 2020 TLA94 DIDVCWDIAADRLFYCHAPM 2021 TLA95 DIIDVCWDIDIDRLYYCEGPW 2022 TLA97 MYDVCWDLQVDKLYFCGDDL 2023 TLA98 DWDVCWDVEVDRLFYCEAPT 2024 TLA99 DFDVCWDLDLDRLVYCDAPM 2025 TLA101 DYDVCWDVHLDRLYYCNSPI 2026 TLA102 DYDVCWDIDEDRLYYCDAFL 2027 TLA103 DYDVCWDLLVDRLYYCDGPT 2028 TLA104 DYDVCWDIYDDRLYYCDTPV 2029 TLA105 ALDVCWDIEEDRLYYCDAPL 2030 TLA106 DYDVCWDVYHDRLFYCEDHL 2031 TLA107 DYDVCWDFEVDRLYYCDGPG 2032 TLA108 EYDVCWDIDVDRLYYCASPM 2033 TLA109 DYDVCWDIELDRLYYCDASI 2034 TLA110 DYDVCWDVELDRLYYCEAPL 2035 TLA111 YYDVCWDLDLDILYYCDAPG 2036 TLA113 DYEVCWDLQMDRLYYCEASI 2037 TLA114 IFDVCWDVDEDRLYYCQAET 2038 TLA115 YTDVCWDIDVDRLYYCDADV 2039 TLA117 EDAVCWDPAHEKLVWCNRFE 2040 TLA118 DYDVCWDVNADRLFYCDDAI 2041 TLA119 DYDVCWDVDEDRLYYCNVPI 2042 TLA120 DYDVCWDLDFDRLYYCHPAE 2043 TLA121 RWDVCWDYHADKLFFCNEDL 2044 TLA122 DYDVCWDVDLDRLYYCEAPM 2045 TLA123 EYEVCWDVHVHRLVFCNVPV 2046 TLA125 KTDVCWDLLLDKLYFCDIDT 2047 TLA126 RDDVCWDYNTDKLYFCGSNI 2048 TLA127 DYDVCWDYDVDRLYYCDFPV 2049 TLA128 NYDVCWDVSEDRLYYCDAPR 2050 TLA129 DWDVCWDLLEDRLYYCEDPM 2051 TLA130 EYDHCWDVSRAKLELCWEHN 2052 TLA131 EYAVCWDEAQQKLVWCKDLN 2053 TLA133 DYDVCWDIYEDRLFYCEMHV 2054 TLA134 KLDVCWDLDVDRLYFCDAPM 2055 TLA135 DYDVCWDVLVDRLYYCDVPM 2056 TLA136 DYDVCWDLHLDRLYYCHAYC 2057 TLA138 DYDVCWDIDLDRLYYCDAGS 2058 TLA139 DYDVCWDIDADRLYYCHAAM 2059 TLA140 KYDVCWDDYLDILYYCNGPL 2060 TLA141 HLDVCWDIDLDHLYYCDLGM 2061 TLA142 MYDVCWDLEVDRLYYCEAPM 2062 TLA143 EYDVCWDIDSDRLYLCVGLM 2063 TLA144 DYDVCWDVHADRLYYCKEDI 2064 TLA145 DYDVCWDLNLDRLYYCDSPM 2065 TLA146 EYDVCWDVQNDRLYYCDAPE 2066 TLA147 DYDVCWDYDVDRLYYCNFQS 2067 TLA148 DYDVCWDYKIDHLYYCDGPV 2068 TLA149 GYDVCWDYEEDRLFYCESPL 2069 TLA150 DYDVCWDLDNDRLFYCDSAM 2070 TLA290 KFHVCWDSHQDKLVYCSLKV 2071 TLA291 LEFVCWDDDRGKLVFCAGHM 2072 TLA293 GHEVCWDMHNEKLVFCRTHI 2073 TLA294 NKAVCWNYIIQEKLEFCNSTM 2074 TLA295 TYAVCWDFNAHKLVFCDDMM 2075 TLA296 EYALCWVEAQEKLVWCKDLI 2076 TLA297 DYAVCWVEDQGKLVWCNGVN 2077 TLA298 DYAVCWDEAQEKLVWCNGLS 2078 TLA299 EYAVCWVHSQEKLDWCTGSK 2079 TLA300 EYAVCWNEARGKLEYCTALK 2080 TLA301 DYAVCWDEHKQKLEFCRTFI 2081 TLA302 EYTVCWDASNEKLYFCHYPL 2082 TLA303 SLDVCWDHHLDKLTFCNLNM 2083 TLA304 QLDVCWDDHYQKLYFCYARI 2084 TLA306 SYDVCWDSRLTKLVYCNASN 2085 TLA307 QYAVCWDEALEKLLWCKELH 2086 TLA308 DYAVCWSEAQDKLEWCTGHN 2087 TLA309 EYAVCWVEEIIEKLLWCEGLN 2088 TLA310 EYSVCWVVDQEKLVWCRSLH 2089 TLA311 EYAVCWSEAQGKLEWCQGSN 2090 TLA312 DYAVCWDVGLEKLVWCKGSL 2091 TLA313 PPAVCWDNNSSKLVFCHSFI 2092 TLA314 SSNVCWDDVLETLRFCHTHM 2093 TLA315 LDQVCWDLYANKLIFCADVI 2094 TLA316 NLDVCWDNNRGKLMFCRNHD 2095 TLA317 SYAVCWNALEEKLVFCTDEI 2096 TLA318 PNEVCWDSHKEKLVFCTHQM 2097 TLA319 EYALCWLEAQEKLDWCTELN 2098 TLA321 ENAVCWDESQEKLVWCNGIR 2099 TLA323 ESDVCWNENVEQLEFCNGRF 2100 TLA324 EYAVCWDEAPEKLIWCEGLN 2101 TLA325 RWAVCWDTDRQKLLFCGPML 2102 TLA326 LDLVCWDENSQKLLFCTKPG 2103 TLA327 QSSVCWDYNSQKLRFCDMSL 2104 TLA328 RLDVCWDEHASKLVFCRSYM 2105 TLA329 ISAVCWDTQLQKLVFCNASM 2106 TLA330 LDTVCWDIHSGRLVFCASLL 2107 TLA331 EYALCWDDPLEKLQWCKDIS 2108 TLA332 EYALCWNEVQEKLVWCKELN 2109 TLA333 EYAVCWDEVQDKLVWCNGVN 2110 TLA334 EYAVCWDEVQERLEWCTRPN 2111 TLA335 EFAVCWDDGQEKLVWCNRLH 2112 TLA336 EYALCWNEVHEQLEFCIGRN 2113 TLA337 RSEVCWDTHLFKLVYCQDWF 2114 TLA338 QSYVCWDEHQSRLRFCSETI 2115 TLA339 PDTVCWDDHLGKLMFCSSIM 2116 TLA340 RNHVCWDEYTEKLRFCGDGA 2117 TLA341 HSDVCWDDHMDKLAFCWHMI 2118 TLA342 FTAVCWDYHSNKLQFCSTLM 2119 TLA343 DYAVCWDDVRGKLVWCKGLK 2120 TLA344 EYDLCWDEIRQKLEWCQGLT 2121 TLA345 EYAVCWDAENEKLEWCEGLN 2122 TLA346 KHFVCWDELNEKLVWCKGLI 2123 TLA347 EYAVCWNEDHAKLEWCQELN 2124 TLA349 LSMVCWDQHQERLVFCINRM 2125 TLA350 AQYVCWDDKEQKLKFCEDQM 2126 TLA351 RYAVCWDESTEKLVYCQNQM 2127 TLA352 NEFVCWDEYKNKLVFCVPTL 2128 TLA353 RFFVCWDHDNSKLVFCSQNM 2129 TLA356 RYAVCWDEAQEKLVWCGRVN 2130 TLA357 AFDVCWVEALEKLEWCLGHN 2131 TLA358 SSDVCWEAHSEKLYFCDPAR 2132 TLA359 KLYVCWDKYKEKLVFCQALM 2133 TLA360 KSEVCWDYDMNKLMFCFYDM 2134 TLA361 LDAVCWDTQQQKLRFCHTHM 2135 TLA362 RSDVCWDAHLDKLIFCAGVM 2136 TLA364 DHAVCWNEFQEKLDWCTGLN 2137 TLA366 QNDVCWDYHRGKLAFCEALI 2138 TLA367 DTDVCWDHVQGKLYFCTKSL 2139 TLA368 TSDVCWNYHYSKLQFCQQSM 2140 TLA369 LQDVCWDLYYQKLYFCHQPM 2141 TLA370 STDVCWDRDHDKLMFCMSVR 2142 TLA371 DYAVCWDEAQGKLGWCIGMN 2143 TLA372 EYAVCWDDAQERLEWCKEPL 2144 TLA373 EYAVCWDESLEKLEWCTGLY 2145 TLA374 EYAVCWDEAQQRLIWCKGLY 2146 TLA375 EYALCWDQDKEILHWCVEFY 2147 TLA376 DNEVCWDGHVDRLVFCDSTM 2148 TLA377 NHDVCWDFHVDRLVYCDTPI 2149 TLA378 DYAVCWDAHMDHLVYCDAIA 2150 TLA379 DYEVCWDDYADRLVYCDLSL 2151 TLA380 SYEVCWDVHLDRLVYCNSVM 2152 TLA381 HYAVCWDFHLDRLVYCDRPS 2153 TLA382 DYEVCWDVIRGRLVYCETPM 2154 TLA383 AYEVCWDVNLDRLVYCEALM 2155 TLA384 GYEVCWDLKWDRLVFCDAPI 2156 TLA385 ALEVCWDFDVDHLVYCAAAL 2157 TLA386 EFEVCWDVHLDRLVYCHATM 2158 TLA388 QYDVCWDIYSDRLVYCDAHL 2159 TLA389 DYEVCWDLYLGRLYYCNAPI 2160 TLA390 EYEVCWDVNVDRLVYCNMPN 2161 TLA391 DYEVCWDVHFDRLVYCDTPL 2162 TLA393 DYEVCWSDDLDRLVYCQALI 2163 TLA394 DDAVCWDYHLDRLVFCETPM 2164 TLA395 DYDVCWDIHLDRLVYCDGPL 2165 TLA396 DYEVCWDVQLDRLVYCDGAM 2166 TLA397 DYEVCWDVHVERLVYCEDLI 2167 TLA398 DYAVCWDVYADRLIYCEVPM 2168 TLA399 DYEVCWDFDIDRLVYCNPPM 2169 TLA402 DYEVCWDVHLDRLVYCDGPL 2170 TLA403 DYSVCWDFYFDRLVYCNALF 2171 TLA404 DYEVCWDVHVGRLIYCNALI 2172 TLA405 SYDVCWDDVVDRLVYCEATL 2173 TLA406 DYEVCWDVEVDRLVYCVGHK 2174 TLA407 PYEVCWDFHLDRLVFCDADK 2175 TLA408 DYEVCWDFLLDREVYCDAPI 2176 TLA409 DWEVCWDVHLDRLVFCETLI 2177 TLA410 DYEVCWDFDFDRLVFCEAVM 2178 TLA411 DYDVCWDVNADRLVYCNATL 2179 TLA412 NIIDVCWEFIILDRLVYCDGPS 2180 TLA413 DYEVCWDVQLEHLVYCEAPI 2181 TLA415 DDQVCWDVKIDRLVYCEALM 2182 TLA416 HSDVCWDFDIDRLVFCDTLI 2183 TLA418 DYEVCWDIHLDRLVYCNTPM 2184 TLA419 DYDVCWDEDVDRLVYCEPPI 2185 TLA420 DYEVCWDFDLDRLVYCDSDV 2186 TLA421 DYEVCWDDHLDRLVYCDAPG 2187 TLA422 DYDVCWDFDLDRLVYCSGPL 2188 TLA423 DYEVCWDDHVDRLVYCDRTL 2189 TLA424 DYEVCWDPHVDRLVYCDAPA 2190 TLA425 IIYEVCWDAIIFDRLVYCEAPI 2191 TLA426 DLEVCWDFDVDRLVYCEAPI 2192 TLA427 GFEVCWDVDLDRLVFCAASE 2193 TLA428 SYAVCWDFDLERLVYCEIPL 2194 TLA429 DHEVCWDFELDRLVYCYKAK 2195 TLA430 KYEVCWDEHIDRLVYCDVPM 2196 TLA431 YSDVCWDFDLDRLVYCEAHL 2197 TLA432 DYEVCWDVNLDRLVYCAGPM 2198 TLA433 HYDVCWDDQVDRLVYCHAPL 2199 TLA434 EYDVCWDFDIDRLVYCVPLS 2200 TLA435 YYEVCWDLNLDRLVYCDVNK 2201 TLA436 NYQVCWDFDLDRLVYCGAHK 2202 TLA437 DYEVCWDVYFGRLVYCDVLM 2203 TLA438 EYVVCWDTEVDHLVYCDAAM 2204 TLA439 YSDVCWDFYYDRLVYCDASI 2205 TLA440 DYEVCWDVDVDRLVYCDGPK 2206 TLA441 DYAVCWDDHVEHLVYCDASW 2207 TLA442 DYDVCWDFDFERLVFCNSSM 2208 TLA443 DYAVCWDDHFDRLVYCDWTT 2209 TLA444 DYDVCWDEHLDRLVYCNGLG 2210 TLA445 EYEVCWDVNVDRLVWCYATM 2211 TLA446 DYEVCWDVIIAERLYYCDAPI 2212 TLA447 DHEVCWDEHVDRLVYCYKAI 2213 TLA448 DYDVCWDDYLDRLVYCEARI 2214 TLA450 HYDVCWDYHLDRLVYCNRHM 2215 TLA451 DYEVCWDIHLDHLVYCDAPW 2216 TLA452 EYDVCWDVDLDRLVYCATTY 2217 TLA453 EYEVCWDFQVDRLVYCMAIT 2218 TLA454 DYEVCWDIIVDRLVYCEAPP 2219 TLA455 YYEVCWDLYLDRLVFCNADL 2220 TLA457 DYEVCWDFREDRLVYCDSIM 2221 TLA458 DYAVCWDFHLNRLVYCDVPV 2222 TLA459 EYDVCWDFQLDRLVYCDSQM 2223 TLA460 DYAVCWDFDEDRLVYCVGPL 2224 TLA461 SERDCNIQKMRKVPCLVML 2225 TLA464 SDWACPMYMIIEIECSVRW 2226 TLA468 VPEVCQWEFVKCLWNA 2227 TLA475 AAWQCLDPEWWVCVMLN 2228 TLA485 LVSSCPYGTPSAVCNGMW 2229 TLA489 GTVYCQPYMSVMECAFRA 2230 TLA491 EDFLCYPAPWVLSAAGT 2231 TLA493 VSGGCPSYMNQWECVFRW 2232 TLA495 HVELCFDKMRYCIMEL 2233 TLA519 TLLACQLKFIKCLNNW 2234 TLA520 LVFLCPGYRAQHHCVLLS 2235 TLA521 QDRVCWDFHLQKLQFCELSM

TABLE 17 EGFR binding MRDs SEQ ID: Ref. No. Peptide Sequence 2252 EGA43 WEFQCHFYDESMFECTLSP 2253 EGA46 WLSMCPHFYHQKCRFWK 2254 EGA47 SRILCMVNPYWMACWLET 2255 EGA49 GMRRCIWEYVMKHQACAPIF 2256 EGA50 HLCGCYLELPGVLLCYCFS 2257 EGA51 ARLLCVVYPGFVACVVQQ 2258 EGA54 VAFRCIIDEMYYMTCWEVE 2259 EGA57 GLIIGCQYVQMFFIIFCAPKT 2260 EGA58 LLMFCHSMWWWPNCYSCV 2261 EGA67 LSLRCTSEMWWGCYLVA 2262 EGA69 EKMYCTYTHFFHMCYLPP 2263 EGA70 LGVICIHWNYCKTFV 2264 EGA78 SVLGCDDVGFYQCLEFL 2265 EGA81 LLILCFHHQLEWKWTCLTME 2266 EGA85 HSGQCTWEVWGRECWYQN 2267 EGA86 QMDTCTWEVWGRDCIQWA 2268 EGA87 DAWGCTWQVWGRECSDPG 2269 EGA88 HEVGCTWEVWGRDCEQIR 2270 EGA90 DPWGCTWEVWGRECPLNL 2271 EGA91 DLHGCTWEIWGRECQIRH 2272 EGA92 DPGGCTWEVWGRECQNSM 2273 EGA93 DMFGCTWEVWGRECPSPQ 2274 EGA95 DAWGCTWEIWGRECARSY 2275 EGA96 TSDSCTWEIWGRECYMQR 2276 EGA98 DQYGCTWEVWGRECVGGA 2277 EGA99 DQYGCIWEQWGRECFVNT 2278 EGA100 DNDGCTWEVWGRECHQFK 2279 EGA101 EYDGCTWEVWGRECHESS 2280 EGA102 GAGNCTWEIWGRDCPQDV 2281 EGA103 TVDRCTWEVWGRECSQNG 2282 EGA104 LADNCTWEVWGRECPITE 2283 EGA105 DNYGCTWEVWGRDCSKSP 2284 EGA106 DAHGCHWEVWGRECTRTA 2285 EGA107 SSDLCTWEIWGRECYSGY 2286 EGA108 DDFGCYWKGWGRECHLPL 2287 EGA109 DWWGCTWEVWGRECLQLE 2288 EGA110 DAHGCTWEVWGRECGQWA 2289 EGA111 DAFGCTWEVWGRECRHVV 2290 EGA112 DAWGCTWEIWGRDCAADN 2291 EGA113 DASGCTWEVWGRECMMWH 2292 EGA114 DPWACTWEVWGRECNHVG 2293 EGA115 ASDNCTWEVWGRECQLWG 2294 EGA116 DMYGCTWEIWGRECHIND 2295 EGA117 DSWGCRWEVWGRECGSDI 2296 EGA118 NPDGCTWEVWGRECYSNR 2297 EGA119 DFFGCTWEIWGRECYWEF 2298 EGA120 SSSFCTWEVWGRDCPQDQ 2299 EGA121 AADRCTWEVWGRDCPQTH 2300 EGA122 LVDLCTWEVWGRECWYSD 2301 EGA124 DSYGCTWEVWGRECPMDT 2302 EGA125 DNWGCTWEVWGRDCHYGT 2303 EGA126 PDFGCTWEVWGRECYGVN 2304 EGA128 QSTQCTWEVWGRECHQMR 2305 EGA129 LPPGCTWEVWGRDCLHPR 2306 EGA130 GEVDCTWEVWGRECYNGW 2307 EGA131 LTVGCTWEVWGRECWQQP 2308 EGA132 DDDGCTWEVWGRECRLPA 2309 EGA133 SLLSCTWEVWGRDCSAFY 2310 EGA134 KDKECTWEVWGRECWQDR 2311 EGA135 DQGGCTWQVWGRDCRSNP 2312 EGA136 DHYGCTWEVWGRECWTVG 2313 EGA137 DAFGCTWAVWGRECRHDQ 2314 EGA138 YGDSCTWEVWGRECMMGR 2315 EGA139 TADPCTWEVWGRECHMRP 2316 EGA140 QDWGCTWEVWGRDCQQYT 2317 EGA141 DSWGCTWEIWGRECWGTE 2318 EGA142 SVDFCTWEVWGRECYWYP 2319 EGA143 AGDGCTWEVWGRECLQES 2320 EGA144 MWPNCTWEVWGRDCAWYH 2321 EGA145 DEYGCSWEVWGRECHTTL 2322 EGA146 DQWGCTWEVWGRECAFNS 2323 EGA147 SSDPCTWEVWGRECPQGP 2324 EGA148 LVLGCWWEVWGRECGSTA 2325 EGA149 KADHCTWEIWGRECAQYT 2326 EGA150 DDWGCSWEVWGRECQIPK 2327 EGA151 PKYGCTWEIWGRECWHST 2328 EGA153 SNLGCTWELWGRDCSLYY 2329 EGA154 DAYGCYWELWGRDCHQIV 2330 EGA155 DYWGCTWEVWGRECTTNP 2331 EGA156 DIWNCTWEVWGRECYLNQ 2332 EGA157 SWSLCTWEVWGRDCYWQK 2333 EGA158 DIWGCTWEVWGRECENYY 2334 EGA159 TNGFCTWEVWGRECWYGS 2335 EGA160 DLYGCTWEIWGRECHITG 2336 EGA161 DENGCTWEVWGRECYIQR 2337 EGA162 DSFGCTWEVWGRECHPQL 2338 EGA163 NHDFCTWEVWGRECWPNA 2339 EGA164 DEHGCTWEVWGRECHRPG 2340 EGA165 DHHGCTWEVWGRDCYWAI 2341 EGA166 WQDNCWWEVWGRECLGDR 2342 EGA167 PTDTCTWEVWGRECYWIR 2343 EGA168 YQDPCTWEIWGRDCHQGM 2344 EGA169 DRWGCTWEIWGRECHQDN 2345 EGA170 DLWGCTWEVWGRECFVVN 2346 EGA171 GLDGCTWEVWGRECYQGS 2347 EGA173 LPIPCYWTTWGKECFMPR 2348 EGA174 LPDPCYWTTWGKECFMFR 2349 EGA175 LADPCYWTTWGKECVIPR 2350 EGA176 LTVPCYWTTWGKECFLPR 2351 EGA177 LPAPCYWTTWGKECFILR 2352 EGA178 LPAPCYWTTWGKECHMPR 2353 EGA179 LPAPCFWTTWGKECFLFR 2354 EGA180 LAAPCYWTTWGKECYMPR 2355 EGA181 PTSPCYWTTWGKECYLLR 2356 EGA182 ITPPCYWTSWGKECHLFR 2357 EGA183 LPAPCYWTTWGKECFMLR 2358 EGA184 LTEPCYWTTWGKECFLPV 2359 EGA185 LPAGCYWTTWGKECFMHN 2360 EGA186 ITSPCYWTTWGKECFIFP 2361 EGA187 LPAPCYWTTWGKECLLFG 2362 EGA188 LPAPCYWTTWGKECFMPQ 2363 EGA189 LPAPCYWTTWGKECFILS 2364 EGA190 LPTPCYWTTWGKECYVYG 2365 EGA192 LPAPCYWTTWGKECYMLP 2366 EGA193 LTPPCYWTTWGKECFMFP 2367 EGA194 LSTQCYWTTWGKECYLFP 2368 EGA195 LPAPCYWTTWGKECLMLR 2369 EGA196 LPYPCYWTTWGKECFMHR 2370 EGA197 LAAPCYWTTWGKECLMIR 2371 EGA198 LTAPCYWTTWGKECYLFH 2372 EGA199 LPAPCYWTTWGKECVLGV 2373 EGA200 LPFPCYWTVWGKECFMPR 2374 EGA202 LSPPCYWTIWGKECHMEQ 2375 EGA203 PNDPCYWTTWGRECLLGR 2376 EGA204 LPPPCYWTTWGKECQMFD 2377 EGA205 LTDPCYWTIWGRECLLPS 2378 EGA206 FNEPCYWTTWGKECYLPR 2379 EGA207 LPAPCYWTNWGKECFMMR 2380 EGA208 FPDPCYWTTWGRECLLPR 2381 EGA209 VTSGCYWTTWGKECFLPR 2382 EGA210 STAACYWTTWGKECYMPR 2383 EGA211 LPGGCYWTTWGKECFMPR 2384 EGA212 IITPCYWTTWGKECYLIP 2385 EGA213 TADPCYWTIWGRECVMSR 2386 EGA215 LADQCYWTTWGKECYLGL 2387 EGA216 LTDPCYWTVWGRECFLLR 2388 EGA217 IDAACYWTTWGKECLLPR 2389 EGA218 LTAPCYWTTWGKECFLLR 2390 EGA219 LPVPCYWTTWGKECLMLR 2391 EGA220 VTDSCYWTTWGRECYLPH 2392 EGA221 LPAGCYWTTWGKECYMFA 2393 EGA222 FTDPCYWTVWGRECLLPR 2394 EGA223 LTAPCYWTTWGKECLILR 2395 EGA224 VTDPCYWTTWGRECLMLP 2396 EGA225 LTSPCYWTTWGKECFMLN 2397 EGA226 LPVPCYWTTWGKECLMFP 2398 EGA227 TADPCYWTTWGRECLMPR 2399 EGA228 LTAPCYWTIWGKECYLLP 2400 EGA229 LAPPCYWTIWGKECLLLR 2401 EGA230 LPSPCYWTTWGKECFMPR 2402 EGA231 VTDGCYWTTWGKECLMPR 2403 EGA233 LPDDCYWTTWGKECYLQR 2404 EGA235 DIDPCWWAPWGRECLMVR 2405 EGA236 TPDPCWWATWGRECILDR 2406 EGA237 LPAPCFWTTWGKECFLLR 2407 EGA238 DNDPCYWTVWGRECLMPL 2408 EGA239 LTAPCYWTTWGKECLLLP 2409 EGA240 LTVPCYWTTWGKECYLLS 2410 EGA241 LIAPCYWTTWGKECYMLP 2411 EGA242 LPEPCYWTTWGRECFLPR 2412 EGA243 LTTPCYWTIWGKECFLPI 2413 EGA244 LPEGCYWTTWGKECFMLR 2414 EGA245 LPAPCYWTTWGKECFILL 2415 EGA247 LKAPCYWTTWGKECYLLP 2416 EGA248 LKDPCYWTTWGRECLMLP 2417 EGA249 VRPGCYWTTWGKECFMPG 2418 EGA250 PSDPCYWTIWGRECYMIP 2419 EGA251 LPAPCYWTVWGKECHMSR 2420 EGA252 LSDPCYWTIWGKECFLIL 2421 EGA254 LTDHCYWTIWGRECLLPR 2422 EGA255 VTDPCYWTTWGRECLLLR 2423 EGA256 LPAGCYWTTWGKECHLHL 2424 EGA257 LPAPCFWTTWGKECYMFT 2425 EGA258 FSDPCYWTTWGRECFWIP 2426 EGA259 LSDPCYWTTWGRECFLLP 2427 EGA260 FKDPCYWTTWGRECYLLP 2428 EGA261 FPDPCYWTTWGRECTLPL 2429 EGA262 LTDPCHWTIWGRECFLPP 2430 EGA263 YDDHCCYLTIWGRECFLPP 2431 EGA265 FSDPCIWTTWGRECFWVH 2432 EGA266 TRDTCYWTVWGRECFLPP 2433 EGA268 HRAPCFSCTACRQPCFSLT 2434 EGA269 FDDPCYWTTWGRECLMLP 2435 EGA270 HNDPCVWTTWGRECFLLP 2436 EGA271 LGDSCFWTTWGRECFLHP 2437 EGA272 FSDPCYWTTWGRECLLIP 2438 EGA273 CTGPCYWTTWGRECCLSYP 2439 EGA274 ASDPCYWTTWGRECFLLP 2440 EGA276 LSDPCYWTTWGRECILLQ 2441 EGA277 LTDPCHWTIWGRECFLLP 2442 EGA278 STDLCYWTIWGRECYLPP 2443 EGA279 FSDPCSWTTWGRECFLPP 2444 EGA280 WRDPCYWATWGRECYLVP 2445 EGA281 LGDPCYWTTWGRECFWLP 2446 EGA282 FRDPCYWTTWGRECFLLP 2447 EGA283 LIDPCYWTTWGRECFLIP 2448 EGA284 LSDNCYWTTWGRECFLPP 2449 EGA285 FTDPCYWTTWGRECFLLP 2450 EGA286 YNDPCYWTTWGRECLLLP 2451 EGA287 SSDPCYWTTWGRECFLLP 2452 EGA288 FGDPCYWTTWGRECYLLRP 2453 EGA289 LTDPCWWTTWGRECLLLP 2454 EGA290 SSDPCYWTTWGRECLLLP 2455 EGA291 LSDPCYWTTWGRECLLLP 2456 EGA292 FRDPCYWTTWGRECFLPP 2457 EGA293 FDDYCCYWATWGRECYLIP 2458 EGA294 YSGPCYWTTWGRECILHP 2459 EGA296 SSDACYWTTWGRECFLLP 2460 EGA297 FSDHCWWSTWGRECYLLP 2461 EGA298 TSDPCYWTTWGRECLLNR 2462 EGA299 TSDPCYWTTWGRECLLLP 2463 EGA300 CSGPCYWTTWGRECCFSLP 2464 EGA302 SIDSCFWTTWGRECFLLP 2465 EGA303 FSDPCYWTTWGRECFLVQ 2466 EGA304 FSDPCYWTTWGRECFETP 2467 EGA305 CRDPCYWTTWGRECYLIP 2468 EGA307 FGDPCYWATWGRECFLIP 2469 EGA308 FADPCYWTTWGRECLLHP 2470 EGA309 FSEPCYWTIWGRECYLLP 2471 EGA310 DDDDCYWTTWGRECFLLP 2472 EGA311 FSDPCTWTTWGRECFLFS 2473 EGA313 FGDPCSWTIWGRECFLLP 2474 EGA314 SKDPCYWTTWGRECFLLP 2475 EGA315 CTGPCYWTTWGRECCFFVP 2476 EGA316 FSDPCYWTTWGRECYLRP 2477 EGA318 YSDPCYWTTWGRECFLMP 2478 EGA319 FGDPCYWTTWGRECFLLP 2479 EGA321 LTDPCYWTTWGRECFLLP 2480 EGA322 YIDTCYWTTWGRECFELP 2481 EGA323 FSDPCFWTTWGRECFWVH 2482 EGA324 FSDHATGQHGVESAFCF 2483 EGA325 FNDPCIWTTWGRECFLLP 2484 EGA326 VSDPCYWTIWGRECFLLP 2485 EGA327 ISDPCYWTTWGRECFLLP 2486 EGA328 FSDPCYWTTWGRECFWVP 2487 EGA329 SRDPCHWTTWGRECLLLP 2488 EGA330 FSEPCYWTIWGRECFLLP 2489 EGA331 VIDTCYWTTWGRECYLLPP 2490 EGA333 ITDPCYWTTWGRECFLRP 2491 EGA335 PSDPCHWTTWGRECFLQS 2492 EGA336 FTDPCYWTIWGRECYLDP 2493 EGA337 FSDHCWWTTWGRECLLLP 2494 EGA338 FSDPCIWTTWGRECFWVP 2495 EGA339 FDDIICCYWTTWGRECFRLP 2496 EGA340 FTDPCYWTIWGRECYEDT 2497 EGA341 SIDPCYWTTWGRECFFLP 2498 EGA342 FADPCYWTIWGRECFLLP 2499 EGA344 FSDPCYWTTWGKECFLLH 2500 EGA346 AFWQCVYTPMHAMCFSHK 2501 EGA347 MTIHWCQPWGCLLLFG 2502 EGA350 LYLSCMTGWWGEIVCVSAS 2503 EGA351 KGLDCVHWPGMHWCWRSD 2504 EGA352 FSDPCYWTIWGRECFLFP 2505 EGA354 FLDPCFWTIWGRECYLLP 2506 EGA355 FTDPCHWTTWGRECIWLP 2507 EGA356 WRAYCHHSPWDFECFQVW 2508 EGA358 VGDPCSWTTWGRECFLFD 2509 EGA359 FSDPCVWTTWGRECLLLP 2510 EGA362 WSPRCFAWIVCLDLS 2511 EGA370 YKWVCMWTPFGHQCVSAA 2512 EGA374 LGDPCYWTTWGRECVLLP 2513 EGA375 KKVFCYYLPFQQYCVTVS 2514 EGA376 FVTWCYAWPDSSACYGLH 2515 EGA377 FSDPCYWTTWGRECFMPP 2516 EGA378 YSDPCYWTTWGRECLLLP 2517 EGA379 WFVTCSFQFEWECVQLH 2518 EGA380 PDSWCHTLYWQLLHCEMSE 2519 EGA381 VDSWCHTLYWQLHYCASQE 2520 EGA382 ADSWCHTLYWSLLHCASSE 2521 EGA383 PDSGCHTLYWNLQHCASLE 2522 EGA384 ADSWCHTLYWQLYYCESLE 2523 EGA386 PESWCHTLYWSLQDCVSSE 2524 EGA387 PDSWCHTLYWQLHHCLSSE 2525 EGA388 ADSWCHTLYWQLLHCVTQE 2526 EGA389 ADSWCHTLYWSLLECESRE 2527 EGA390 PDSWCHTLFWDLQHCVSGE 2528 EGA391 PDSLCHTLYWSLRHCEIEE 2529 EGA392 PDSLCHTLYWQLQHCAFWE 2530 EGA393 ADSWCIITLYWQLYYCEVWE 2531 EGA394 AESWCHTLYWSLHHCVYSE 2532 EGA395 TDSWCHTLYWNLLHCASYE 2533 EGA396 PDSWCHTLYWQLLHCATLE 2534 EGA397 PDSWCHTLYWLLHECESRE 2535 EGA398 PDSWCHTLYWSLQFCESSE 2536 EGA399 PDSWCHTLYWQLHHCEILE 2537 EGA400 PDSWCHTLYWKLHHCLSME 2538 EGA401 VDSWCHTLYWQLLHCAMSE 2539 EGA402 AESWCHTLYWQLVHCTSLE 2540 EGA403 PDSWCHTLYWDLLHCESSE 2541 EGA404 ADSWCIITLWWQLIIIICLFEE 2542 EGA405 TDSWCHTLYWLLHDCTSRE 2543 EGA407 ADSWCHTLYWQLLHCVAQE 2544 EGA408 PDSSCHTLYWLLEHCLSVE 2545 EGA409 PDSWCHTLYWQLHYCTILE 2546 EGA410 PDSWCHTLYWQLLHCEISE 2547 EGA411 PDSWCHTLYWQLLHCAASE 2548 EGA412 PDSWCHTLYWQLHHCVSWE 2549 EGA413 TDSWCHTLYWALQHCVSTE 2550 EGA414 PDSWCHTLYWQLLHCTTQE 2551 EGA415 ADSWCHTLYWQLLHCISLE 2552 EGA416 ADSWCHTLYWQLQHCLSQE 2553 EGA417 PDSYCHTLYWSLIHCARSE 2554 EGA418 TDSWCHTLYWELQYCLMQE 2555 EGA419 TDSWCHTLYWQLHHCAILE 2556 EGA420 PDSWCHTLYWQLYHCVSLE 2557 EGA421 ADSWCHTLYWQLLHCVNQE 2558 EGA422 PDSWCHTLYWSLLHCARQE 2559 EGA423 ADSWCHTLYWALQHCARSE 2560 EGA424 ADSWCHTLYWSLLHCASED 2561 EGA425 ADSWCHTLYWDLLHCANLE 2562 EGA426 ADSWCIITLYWQLLIICESSE 2563 EGA427 PDSWCHTLYWDLLHCPAGE 2564 EGA428 ADSWCHTLYWQLHHCTSLE 2565 EGA429 ADSWCHTLYWSLQHCASSE 2566 EGA430 PDSWCHTLYWQLYHCEMSE 2567 EGA431 PDSWCHTLYWDLMHCHHEE 2568 EGA432 PDSWCHTLYWQLYYCATSE 2569 EGA434 ADSWCHTLYWQLLHCASGE 2570 EGA435 ADSWCHTLYWQLYHCAYWE 2571 EGA436 TDSWCHTLYWSLAHCAFLE 2572 EGA437 ADSWCHTLYWQLLHCETLE 2573 EGA438 ADSWCHTLYWQLHHCVSLE 2574 EGA439 ADSWCHTLYWDLLHCESLE 2575 EGA440 ADSWCHTLYWQLLHCEFSE 2576 EGA441 TDSWCIITLYWNLVIICASQD 2577 EGA442 PDSWCHTLYWDLLYCVNQE 2578 EGA443 PDSWCHTLYWNLHHCLSME 2579 EGA444 ADSWCHTLYWALHHCSIQE 2580 EGA445 TDSWCHTLYWALQHCVAQE 2581 EGA446 PDSRCHTLYWALRYCAYQE 2582 EGA447 ADSWCHTLYWNLLYCAIRE 2583 EGA448 VDSFCHTLYWQLIHCPIEE 2584 EGA449 ADSWCHTLYWNLLHCRALE 2585 EGA450 PDSWCHTLYWQLLDCATRE 2586 EGA451 ADSWCHTLYWNLRHCEIQE 2587 EGA452 PNSWCIITLYWDLLIICVSRE 2588 EGA453 ADSWCHTLYWQLHHCAISE 2589 EGA454 ADSWCHTLYWSLLHCESFD 2590 EGA455 ADSWCHTLYWQLQHCVSWE 2591 EGA456 ADSWCHTLYWQLQHCISYE 2592 EGA457 PDSWCHTLYWSLQHCAIYE 2593 EGA458 PDSWCHTLYWQLLDCTSSE 2594 EGA459 PDSWCHTLYWHLQHCLSQE 2595 EGA460 AESWCHTLYWSLYHCASEE 2596 EGA461 PDSWCHTLYWQLQHCVSWE 2597 EGA462 ADSWCHTLYWQLLHCVSQE 2598 EGA463 ADSWCHTLYWQLQYCVMQE 2599 EGA465 ADSWCHTLYWQLLHCESLE 2600 EGA468 ADSWCHTLYWQLHHCVLEE 2601 EGA469 PDSWCHTLYWSLHHCLISE 2602 EGA470 PDSWCHTLYWQLIHCERQE 2603 EGA471 PDSWCHTLYWNLQHCERME 2604 EGA472 ADSWCHTLYWQLKHCEFTE 2605 EGA473 ADSWCHTLYWNLLYCANQE 2606 EGA474 PDSWCHTLYWSLQHCLNME 2607 EGA475 PDSWCHTLYWSLYHCTISD 2608 EGA476 ADSWCIITLWWQLLIICESGE 2609 EGA477 SDSWCHTLYWSLRHCESLE 2610 EGA478 ADSWCHTLYWDLQHCLRWE 2611 EGA479 PDSWCHTLYWQLLYCESWE 2612 EGA480 ANSWCHTLFWDLHHCLSLE 2613 EGA481 ADSWCHTLYWQLYHCTSLE 2614 EGA482 PDSWCHTLYWQLRHCEISE 2615 EGA484 PDSGCHTLYWSLQHCTFWE 2616 EGA485 ADSWCHTLYWQLYHCEVLE 2617 EGA486 PDSWCHTLYWDLHWCVSLE 2618 EGA487 ADSWCHTLYWSLLHCAIGE 2619 EGA489 ADSWCHTLYWQLHHCLWLE 2620 EGA490 ADSWCHTLYWSLAHCLSGE 2621 EGA491 AESWCHTLYWDLHYCVYHE 2622 EGA492 SDSWCIITLYWSLQIICVISE 2623 EGA493 ADSWCHTLYWNLLHCEYRE 2624 EGA494 ADSWCHTLYWQLLHCASEE 2625 EGA495 PESWCHTLYWQMIFCESFE 2626 EGA496 ANSWCHTLYWQLHHCEILE 2627 EGA497 PDSWCHTLYWALHHCASSE 2628 EGA498 PDSSCHTLYWQLHHCARRE 2629 EGA499 ADSWCHTLYWDLQHCLILE 2630 EGA500 ADSWCHTLFWNLKHCLIQE 2631 EGA502 ANSWCHTLYWDLMYCVWQE 2632 EGA503 ADSWCHTLYWQLHHCVTLE 2633 EGA504 PDSWCIITLYWALIIIICVSSE 2634 EGA505 GGSGCHGYLWQLLHCETWEG QAGQAAAGGSTDSWCHTLYW QLYHCASSE 2635 EGA506 PESWCHTLYWNLQHCLSGE 2636 EGA507 PDSWCHTLYWQLTHCESLE 2637 EGA509 PDSWCIITLYWVLLDCARIIE 2638 EGA510 PDSWCHTMYWSLIHCMNME 2639 EGA511 AASWCQALYWHQFCCASWE 2640 EGA514 ADSWCHTLFWQLQHCVWQE 2641 EGA515 TDSWCHTLYWSLLHCESLG 2642 EGA517 PDSWCHTLYWQLYHCLSLE 2643 EGA519 SPDSCWWAIWGKECILLP 2644 EGA520 LSSGCYWAVWGRECLLIP 2645 EGA521 LADGCWWAAWGRECLLPP 2646 EGA522 LPGGCWWSAWGRECILLP 2647 EGA523 LPEGCWWAVWGRECQLQP 2648 EGA524 LPEGCWWSTWGRECLLLP 2649 EGA526 LPEGCYWAIWGKECFLPP 2650 EGA527 LPDGCWWAQWGRECLLLP 2651 EGA528 DPDGCHWEVWGRECILLP 2652 EGA529 LPEGCWWATWGRECLLPP 2653 EGA530 LPDGCWWSLWGRECFLPP 2654 EGA531 LLDVCWWSTWGRECLLQP 2655 EGA532 LPEGCWWAHWGRECVMVP 2656 EGA533 LPDDCYWAQWGRECHMLP 2657 EGA534 LPEGCYWAVWGRECILPR 2658 EGA535 VPEGCWWSPWGRECLLPP 2659 EGA536 LPSGCWWAVWGKECLLLP 2660 EGA537 MPEGCWWSLWGRECMLLP 2661 EGA538 LPEGCWWATWGKECLLLP 2662 EGA539 LPEGCYWAIWGKECYLQA 2663 EGA540 LPEGCWWATWGRECFLLP 2664 EGA541 PPDGCWWAVWGRECLLLP 2665 EGA542 LPESCWWSPWGRECLLPP 2666 EGA543 LSEGCWWAVWGKECTLPP 2667 EGA544 LSEGCWWAVWGRECLLQP 2668 EGA545 LPVGCWWSVWGRECLLPP 2669 EGA546 VPAGCWWAPWGRECILSP 2670 EGA547 LPSACNWSPWSWQCFWLP 2671 EGA548 LPEGCWWAVWGKECVLSP 2672 EGA550 LPEGCWWSSWGKECFLHP 2673 EGA551 LPDGCWWSPWGRECILLP 2674 EGA552 LPDGCWWAVWGRECILPP 2675 EGA553 LPEGCWWATWGKECVLLP 2676 EGA555 LPDGCWWSAWGKECFLLP 2677 EGA556 LPEGCWWSVWGKECLLLP 2678 EGA557 LPEGCWWAIIWGRECLLQP 2679 EGA558 FPERCWWAMWGKECYLLP 2680 EGA559 LPEGCWWTVWGKECYLLP 2681 EGA560 LSADCWWSPWGRECILKP 2682 EGA561 PSQGCWWSVWGRECFLPP 2683 EGA562 FPEECWWATWGRECFLPR 2684 EGA563 FPEGCWWATWGKECLLRP 2685 EGA564 LPDGCWWALWGKECFLMH 2686 EGA565 LPEGCYWAQWGKECFLVP 2687 EGA566 LADSCYWTIWGRECLLPP 2688 EGA567 FPADCWWSTWGRECFLQP 2689 EGA568 LPEGCYWAVWGRECYLVP 2690 EGA569 LPEGCWWAVWGRECLLLP 2691 EGA570 DPEGCFWAVWGRECLLQP 2692 EGA571 FADGCWWEVWGRECLLLP 2693 EGA572 DPEGCWWAVWGRECLLRP 2694 EGA573 VSEGCWWAIWGRECILLP 2695 EGA574 LPEGCWWSPWGKECLMTP 2696 EGA575 LPDGCWWALWGKECILRP 2697 EGA576 LPDGCWWSQWGRECILVP 2698 EGA577 LPQDCWWAIWGRECFMLP 2699 EGA578 LPDVCWWAVWGRECLLMP 2700 EGA579 LPEGCWWAKWGKECVLPS 2701 EGA580 LPEGCYWTIWGRECFLFP 2702 EGA581 LPESCWWAVWGRECLLSP 2703 EGA582 SPDGCWWSQWGRECIMLP 2704 EGA583 KPESCWWANWGRECLLLP 2705 EGA584 LSEGCWWATWGKECLLLP 2706 EGA585 VAGGCWWATWGRECLLPP 2707 EGA586 SAEGCWWAVWGRECLLLP 2708 EGA587 LPEGCYWAIWGRECLLPR 2709 EGA588 LPEGCFWAVWGKECFLLP 2710 EGA589 LTEGCWWAQWGRECILLP 2711 EGA590 LPKGCFWTTWGKECLLLP 2712 EGA591 LPEGCWWTVWGRECLLLP 2713 EGA592 LPDDCWWAPWGRECFLRP 2714 EGA594 DSDGCYWAVWGRECFFLP 2715 EGA595 LRDGCWWAIWGRKCLLLP 2716 EGA596 VDEGCWWAVWGRECVLPE 2717 EGA597 HTDGCWWAQWGRECLLVP 2718 EGA598 LPEGCWWAPWGKECLLLP 2719 EGA599 LPDGCWWASWGRECFLIP 2720 EGA600 LPEGCWWAVWGRECILLS 2721 EGA601 LPDACWWSVWGKECILEP 2722 EGA602 DPHGCWWSPWGRECVLLD 2723 EGA603 LPQGCYWAVWGRECLLPL 2724 EGA604 LDAPCYWTVWGKECFLFS 2725 EGA605 DHYGCYWTQWGRECFPMY 2726 EGA606 LPEGCYWAVWGKECFLLP 2727 EGA607 LHEGCWWAVWGKECFLLP 2728 EGA608 LPEGCWWSKWGRECILVP 2729 EGA609 LPQGCWWANWGRECFLLP 2730 EGA610 LPEGCWWATWGRECILLP 2731 EGA611 ISDGCWWAIWGRECLLMP 2732 EGA612 TPDGCYWAVWGRECFLRP 2733 EGA613 LPGDCWWAIWGRECLLPP 2734 EGA614 FADGCWWAVWGRECFWEP 2735 EGA615 LPEGCWWAVWGKECFMLP 2736 EGA616 LPEGCWWSTWGKECVLLP 2737 EGA617 LPDGCWWSLWGKECHLLQ 2738 EGA618 LPEGCWWAHWGKECQLRP 2739 EGA619 DPEGCWWATWGRECLLLP 2740 EGA620 LPDGCWWAHWGRECLLPP 2741 EGA621 LPDGCWWAKWGKECLLWP 2742 EGA622 LPDGCWWSVWGKECILLP 2743 EGA624 RSEGCWWATWGRECFLHP 2744 EGA627 LPEVCWWATWGHECLLLP 2745 EGA628 LPEGCWWSSWGRECFLLP 2746 EGA629 LPEGCYWATWGKECLLQP 2747 EGA630 LPQGCWWATWGRECLLRP 2748 EGA631 LPDGCWWAVWGRECLLLP 2749 EGA632 VSEGCWWAIWGRECFLQP 2750 EGA633 VADGCVWMVWGRECLLLP 2751 EGA634 VPEACWWALWGRECILRP 2752 EGA635 LPQGCYWTVWGRECLLVP 2753 EGA636 QPEYCWWATWGRECLLVP 2754 EGA637 LPEGCFWAIWGSECLLLP 2755 EGA638 LPEACWWSTWGRECLLLP 2756 EGA639 VPEGCWWAVWGVECQLLP 2757 EGA640 LPESCCWTDWGHECFLPP 2758 EGA641 FPEDCWWSVWGRECLLLP 2759 EGA642 LSGGCWWAVWGRECLLLP 2760 EGA643 LPDGCWWSQWGRECILLP 2761 EGA644 MPEGCWWAAWGRECILLP 2762 EGA645 LPEGCYWAVWGRECLLLP 2763 EGA646 LPDGCYWAIWGRECILPN 2764 EGA647 LPEGCWWAIWGRECLLLP 2765 EGA648 LPEGCWWATWGRECLLLP 2766 EGA650 LPEGCYWTTWGKECLLRP 2767 EGA651 LPDGCYWAVWGKECYQIR 2768 EGA652 LPEGCWWSSWGKECFLLP 2769 EGA654 LPEGCWWAHWGKECLLGP 2770 EGA655 IPDGCWWTVWGRECFLIIP 2771 EGA656 LPEGCWWSIWGRECILLP 2772 EGA657 LPEGCHWTMWGKECFLPP 2773 EGA658 LPEGCWWATWGRECILPP 2774 EGA659 LPEGCWWAPWGKECILPH 2775 EGA660 LPEGCWWSNWGRECFMRQ 2776 EGA661 LPSGCWWAIWGRECLLPP 2777 EGA662 LADGCYWAIWGRECLLLP 2778 EGA663 LPEGCFWTVWGKECLLLP 2779 EGA664 LPEGCYWTTWGRECLLLP 2780 EGA665 LPDGCWWSTWGRECVLLP 2781 EGA666 MPDNCWWALWGKECFLMP 2782 EGA667 LPEGCWWAVWGKECLLLP 2783 EGA668 DPYGCWWTQWGRECHLLP 2784 EGA669 LPEGCYWALWGKECLLPP 2785 EGA670 GPEDCWWSTWGRECYLAP 2786 EGA671 LPEGCIWTTWGKECFLQF 2787 EGA672 LPAGCWWSAWGRECLLTP 2788 EGA674 LNEQCWWAVWGHECLLLP 2789 EGA675 IPDDCWWAVWGRECLLLN 2790 EGA676 LPEGCWWSQWGRECILPP 2791 EGA677 LPEGCFWTIWGQECFLLP 2792 EGA678 LPDGCWWSQWGKECILLP 2793 EGA679 LPEGCWWSTWGRECILLP 2794 EGA680 LPDGCWWSIWGRECLLAP 2795 EGA681 VPDGCWWSPWGRECLLPP 2796 EGA682 VPESCWWAVWGRECLLLP 2797 EGA683 LPDGCWWAMWGRECILEP 2798 EGA684 LPEGCYWTIWGKECFLLP 2799 EGA685 LPDNCVWMVWGRECLLVS 2800 EGA686 LTEGCWWATWGRECFLLP 2801 EGA687 LPKGCFWTVWGKDCYLLP 2802 EGA688 LPEGCFWAKWGKECYLTQ 2803 EGA689 LPDGCWWAVWGRECLMLP 2804 EGA693 DSYGCFWTIWGRECLLLP 2805 EGA694 LSDGCWWAVWGRECLIQK 2806 EGA695 HPEGCWWAVWGKECLLPP 2807 EGA696 LPEGCWWAIWGRECLMQP 2808 EGA697 SLEGCWWSPWGKECFLPP 2809 EGA698 LPSGCWWSTWGRECLLLP 2810 EGA699 LPDGCYWAVWGRECLLLP 2811 EGA700 LPEACWWATWGRECILLP 2812 EGA701 LPEGCCWTVWGLECFLCP 2813 EGA702 MPEGCYWAQWGKECLLVP 2814 EGA703 LPEGCLWMVWGKECFLVP 2815 EGA704 LPEGCHWAVWGKECQLQP 2816 EGA705 LPEGCWWAPWGRECILPP 2817 EGA706 LPEGCHWEIWGKECFLPP 2818 EGA707 LPKGCYWATWGRECLLVP 2819 EGA708 LPEGCYWAVWGKECLLRP 2820 EGA710 IGDACYWTVWGRECLLLP 2821 EGA711 LPDGCVWTVWGRECFGFP 2822 EGA712 SPDGCYWALWGRECLLAP 2823 EGA713 LPQGCWWSVWGKECILRP 2824 EGA714 LPKGCWWAVWGKECLMQS 2825 EGA715 LPEGCWWAVWGRECLMLH 2826 EGA716 LPEGCYWTTWGKECFLLP 2827 EGA717 YAEHCSMFPNDWICTLVT 2828 EGA718 YAEHCWQFPTDWICTLMP 2829 EGA719 YVEHCWQFPTDWICTLKT 2830 EGA720 YVDHCWHFPADWICGLST 2831 EGA721 YEEIICRLFPADWICSLLQ 2832 EGA722 YLEHCRHFPTDWICSLLP 2833 EGA723 YLEHCRQFPGDWICTLLE 2834 EGA724 YAEHCRMFPTDWICSLIT 2835 EGA725 YVEHCCKFPTDWICTLNS 2836 EGA726 YAEHCWLFPTDWICGLRA 2837 EGA727 YAEHCRLFPTDWICTLLT 2838 EGA728 YEEHCWLFPTDWICTLLT 2839 EGA731 YAEHCWQFPTDWICSLSS 2840 EGA732 YAAHCWQFPTDWICTLLP 2841 EGA733 YAEHCSLFPSDWICSLMT 2842 EGA734 YAEHCSQFPCDWICTLLT 2843 EGA735 YAEHCCQFPSDWICTLMS 2844 EGA736 YAEHCRLFPCDWICTLMT 2845 EGA737 YAEHCWLFPTDWICTLLP 2846 EGA738 YAEHCWMFPSDWICSLPT 2847 EGA739 YADHCEMFPNDWICTLRT 2848 EGA740 YAEHCRMFPSDWICTLIP 2849 EGA741 YAEHCRLFPTDWICTLTT 2850 EGA742 YAEHCSQFPTDWICTLIT 2851 EGA743 YEEHCWLFPNDWICSLMT 2852 EGA744 YAEHCQLFPSDWICSLET 2853 EGA746 YAEHCAQFPTDWICSLIS 2854 EGA747 YAQHCYQFPSDWICSLLP 2855 EGA748 YAEHCRHFWTDWICSLKT 2856 EGA749 YGEHCRSFPTDWICTLLP 2857 EGA750 YSTHCRLFPTDWICTLEG 2858 EGA751 YAEHCWHFPTDWICSLLP 2859 EGA752 YFEHCRQFPTDWICTLMP 2860 EGA753 YAEHCRLFPTDWICTIKS 2861 EGA755 YVEIICWQFPSDWICTLLP 2862 EGA757 YAEHCWRFPGDWICSLIT 2863 EGA758 YAEHCRLFPTDWICSLRE 2864 EGA759 YAEHCSMFPTDWICTLVT 2865 EGA760 YLQHCLLFPTDWICTLLP 2866 EGA761 YAEHCTLFPTDWICTLLP 2867 EGA762 YAEHCWMFPTDWICSLRS 2868 EGA763 YAEHCWLFPKDWICTLIT 2869 EGA764 YEEHCWQFPGDWICSLRG 2870 EGA765 YAEHCRIFPTDWICTLST 2871 EGA766 YAEHCWQFPSDWICSLIE 2872 EGA767 YAEHCSHFWTDWICTLST 2873 EGA768 YAEHCRQFPSDWICTLLS 2874 EGA769 YAAHCSLFPTDWICTLMP 2875 EGA770 YAEHCWLFPKDWICTLDM 2876 EGA773 YAEHCLQFPADWICTLRT 2877 EGA774 YAGIICALFPSDWICSLLP 2878 EGA775 YAEHCSYFPTDWICTLRT 2879 EGA776 YLDHCRLFPTDWICSLLP 2880 EGA777 YVEHCRLFPCDWICSLMI 2881 EGA779 YAEHCFQFPHDWICSLMT 2882 EGA780 YQEHCWRFPNDWICSLLP 2883 EGA781 YAEHCRLFPGDWICTLST 2884 EGA782 YAEHCSMFPSDWICSLMT 2885 EGA783 YAEHCWHFPTDWICTLRS 2886 EGA784 YAEHCGLFPGDWICTLKN 2887 EGA785 YAEHCNMFPTDWICTLME 2888 EGA786 YAEHCSFFPADWICTLIP 2889 EGA788 YAEHCWMFPTDWICSLNR 2890 EGA789 YPEHCWQFPGDWICTLLT 2891 EGA790 YADHCRLFPTDWICTLVT 2892 EGA791 YAEHCRLFPSDWICSLVT 2893 EGA792 YAEHCRQFPADWICTLMT 2894 EGA793 YSEHCWMFPSDWICTLME 2895 EGA795 YAEHCSQFPTDWICTLLT 2896 EGA796 LAEHCYLFPCDWICSLLT 2897 EGA798 YAEHCWQFPADWICTLLP 2898 EGA799 YREHCSLFPNDWICTLMP 2899 EGA801 YAEHCSLFPTDWICSLMP 2900 EGA802 YEEHCWLFPSDWICSLRQ 2901 EGA804 LPEPCWWAPWGRECLLLP 2902 EGA805 LPQGCWWAVWGKECHLLP 2903 EGA806 LYDTCYWTTWGKECLMPR 2904 EGA807 LPDGCTWMVWGRECFLLP 2905 EGA808 LPGGCWWAVWGRECQLLP 2906 EGA809 HPDDCWWALWGRECLLLP 2907 EGA810 KCNFNPWKAACG 2908 EGA811 LPEGCWWALWGRECLLLP 2909 EGA812 LLDACWWATWGKECIWRN 2910 EGA813 LTYVCWWNMWGYECLPYE 2911 EGA814 LPDGCWWAQWGRECLLKP 2912 EGA815 LPAPCYWTTWGKECFIVP 2913 EGA816 LIAPCYWTIWGKECFILD 2914 EGA817 HPDNCWWAVWGRECLLLP 2915 EGA818 LSDPCYWTTWGKECFLLR 2916 EGA819 LTAPCYWTTWGKECLLLR 2917 EGA820 LAEGCIWTTWGRECFLLP 2918 EGA821 LPDGCYWTVWGRECFLPP 2919 EGA822 LRDPCWWAVWGRECILLP 2920 EGA823 QSPGCWWAVWGVECILLP 2921 EGA824 IHEGCWWSSWGKECLLLP 2922 EGA825 LPEGCWWATWGRECLLEP 2923 EGA826 LPAGCYWANWGKECFLVP 2924 EGA828 LTDPCYWTTWGKECLLHR 2925 EGA830 LPEGCWWAPWGKECLLSP 2926 EGA831 LPAPCYWTTWGKECLMPR 2927 EGA832 LPDGCYWSMWGRECFLVP 2928 EGA833 VPQGCYWATWGRECYLLP 2929 EGA834 LPDSCWWSTWGRECILLP 2930 EGA835 LPAPCYWTTWGKECLLQH 2931 EGA836 LQEGCFWAVWGKECILLP 2932 EGA837 LNEHCYWALWGKECFLPR 2933 EGA838 LPEGCWWSPWGRECLLLP 2934 EGA840 LPEGCFWTVWGKECLLMP 2935 EGA841 LPDGCYWTTWGKECFMPR 2936 EGA842 LPEGCWWSPWGKECLLMP 2937 EGA843 LTPPCYWTIWGKECFMRQ 2938 EGA844 LPEGCYWSVWGKECLLES 2939 EGA846 LTDQCYWATWGRECILLR 2940 EGA847 FPDGCYWAIWGRECLLQP 2941 EGA848 LSGGCYWTTWGRECILLP 2942 EGA849 LSSPCYWTTWGKECFIIR 2943 EGA850 FPEGCWWALWGKECFLQP 2944 EGA851 LPDGCWWSVWGRECLMLP 2945 EGA852 LPEDCWWAIWGRECLLTP 2946 EGA853 LPEPCYWTTWGKECFMIH 2947 EGA854 LPQGCYWAVWGRECLLLP 2948 EGA855 LPAGCWWSLWGKECYLLP 2949 EGA856 LPEECWWAIWGRECHLLP 2950 EGA857 LPAGCWWSVWGRECLLMP 2951 EGA858 MPEGCFWAIWGRECFLLP 2952 EGA859 LPDPCWWAPWGRECVLMP 2953 EGA860 IPDGCYWAIWGRECFLLP 2954 EGA861 LPAPCYWTTWGKECFLLR 2955 EGA862 LTDSCYWTTWGRECFLIP 2956 EGA863 IHDPCYWTVWGRECLLLP 2957 EGA864 LPEGCWWSAWGRECILLQ 2958 EGA865 LMGDCWWTVWGKECLLQP 2959 EGA868 VPDSCWWSQWGRECLLPP 2960 EGA869 LPEGCWWSVWGKECLMLP 2961 EGA870 LATPCYWTTWGKECLIPR 2962 EGA871 LLLGCSWHFWVYECLPPP 2963 EGA872 INPPCYWTTWGKECFLHR 2964 EGA873 LPEGCWWTQWGKECHLAP 2965 EGA874 FPAGCYWAHWGRECLLLP 2966 EGA875 LPEGCWWSIWGKECLLLP 2967 EGA876 LPEGCYWAIWGKECHMTP 2968 EGA878 IPEGCWWQPWALECFLLP 2969 EGA879 IAEGCWWSIWGKECFLLP 2970 EGA880 LPEPCYWTTWGKECHLPN 2971 EGA881 LPPRCLWNPWGPECFLIH 2972 EGA882 LTAPCWWTSWGRECYLFG 2973 EGA883 RPDSCYWTTWGRECLWPR 2974 EGA884 LPERCWWAIWGRECILLP 2975 EGA885 ISQGCWWSKWGRECLLPP 2976 EGA886 IPHGCWWAIWGRECLLLP 2977 EGA888 YPEHCWWFPSDWICTLMT 2978 EGA889 YAEHCWRFPSDWICTLMT 2979 EGA890 YADHCRLFPNDWICTLIT 2980 EGA891 YAQHCYLFPMDWICTLTT 2981 EGA892 YSEHCWQFPDDWICTLMT 2982 EGA893 YAEHCRVFPTDWICTLRP 2983 EGA894 YGQHCSLFPTDWICSLMT 2984 EGA895 YAEHCRQFRTDWICTLMT 2985 EGA896 YAEHCLTFPTDWICSLAQ 2986 EGA897 YAGHCRDFPSDWICSLVP 2987 EGA898 YAEHCWYFPTDWICTLT 2988 EGA899 YQEHCWLFPTDWICSLTS 2989 EGA900 YADHCCHFPADWICTLNA 2990 EGA901 YAEHCWLFPTDWICSLMP 2991 EGA902 YADHCLQFPLDWICTLMK 2992 EGA903 YAEHCTQFPTDWICTLTS 2993 EGA904 YAEHCRQFPSDWICTLMT 2994 EGA905 YSEHCYLFPTDWICTLPT 2995 EGA906 YVDHCWDFPTDWICTLLS 2996 EGA907 YIEHCWQFPTDWICTLMT 2997 EGA908 YADHCRFFPTDWICTLMP 2998 EGA909 YAQHCMQFPTDWICTLMT 2999 EGA910 YADIICWQFPSDWICTLLA 3000 EGA911 YARHCMQFPTDWICSLTT 3001 EGA912 YAEHCWQFPTDWICTLRS 3002 EGA913 YEDHCSLFPKDWICGLMP 3003 EGA914 YAEHCWQFPTDWICTLLP 3004 EGA915 YEEHCQLFPTDWICTIRT 3005 EGA916 YAEHCWLFPTDWICSLMT 3006 EGA918 YAEHCWQFPTDWICTLMS 3007 EGA919 YAEHCRQFPTDWICGLMT 3008 EGA920 YADHCSQFPTDWICTLIP 3009 EGA921 YSEHCWLFPTDWICTLVP 3010 EGA922 YAAHCWQFPTDWICTLIP 3011 EGA924 YAEHCWLFPSDWICTLMP 3012 EGA925 YAEHCYMFPTDWICTLTA 3013 EGA927 YTEHCWLFPTDWICSLMP 3014 EGA929 YEQHCWHFPNDWICTLMT 3015 EGA930 YAEIICWLFPTDWICTLMT 3016 EGA931 YYEHCTLFPSDWICTLMT 3017 EGA932 YEEHCALFPTDWICTLH 3018 EGA933 YGEHCWQFPTDWICTLVT 3019 EGA934 YSEHCWLFPSDWICSLDT 3020 EGA935 YDEHCRQFPTDWICKLTT 3021 EGA936 YAEHCYRFPTDWICTLMS 3022 EGA937 YAGHCSRFPTDWICTLMA 3023 EGA938 YAQHCWQFPADWICTLIE 3024 EGA939 YDEHCLKFPADWICSLPR 3025 EGA940 YAGHCNLFPTDWICTLKT 3026 EGA941 YEEHCGLFPTDWICTLMD 3027 EGA942 YSEHCWLFPTDWICTLPT 3028 EGA944 YAEHCWMFPTDWICSLLP 3029 EGA945 YAEHCWQFPSDWICTLLP 3030 EGA946 YAEHCWRFPTDWICTLMT 3031 EGA947 YTSHCRHFPNDWICTLLP 3032 EGA948 YAEHCWQFPHDWICTLTK 3033 EGA949 YADHCWLFPNDWICTLIK 3034 EGA950 YAEHCWLFPTDWICTLRT 3035 EGA951 FQGHCALFPTDWICTLKS 3036 EGA952 YAEHCRLFPADWICSLMT 3037 EGA953 YAEHCGLFPTDWICTLMT 3038 EGA954 YREHCGQFPNDWICSLLT 3039 EGA955 YVEHCYQFPADWICTLKT 3040 EGA956 YAEHCRHFPSDWICTLTK 3041 EGA957 YAEHCSQFPTDWICSLLT 3042 EGA959 YAVHCWQFPADWICSLEM 3043 EGA960 YEEHCLQFPADWICTLLP 3044 EGA961 YAEHCGMFPNDWICSLTK 3045 EGA962 YADIICAIIFPTDWICTLMT 3046 EGA963 YPEHCWLFPTDWICTLVA 3047 EGA964 YAEHCWQFPADWICSLPS 3048 EGA965 YAEHCWQFPTDWICSLLD 3049 EGA966 YAQHCWMFPADWICTLMT 3050 EGA967 YLAHCWHFPNDWICSLLP 3051 EGA968 YTKHCQRLPTYKICNLFM 3052 EGA969 YSEHCWLFPTDWICHLMP 3053 EGA970 YAEHCYNFPSDWICSLLT 3054 EGA971 YAEHCWQFPTDWICSLMT 3055 EGA972 YEEHCWQFPADWICTLLT 3056 EGA973 YAEHCWHFPSDWICTLKE 3057 EGA974 YAMHCSQFPSDWICTLMT 3058 EGA975 YAEHCRQFPTDWICSLMT 3059 EGA976 YEEHCWLFPSDWICTLMP 3060 EGA977 YAEHCWLFPTDWICNLIP 3061 EGA978 YEKIICYQFPSDWICSLKT 3062 EGA979 LPDGCCWLQWGMECHLCP 3063 EGA980 IPDDCYWTVWGRECLLVP 3064 EGA981 LTDGCYWTIWGRECLLLP 3065 EGA982 LPDGCWWSTWGRECLLVP 3066 EGA984 LPEGCYWAIWGKECLLPL 3067 EGA985 DPYGCYWAAWGRECLLLP 3068 EGA986 LPEGCWWSVWGRECLLPS 3069 EGA987 LPEDCWWATWGRECLLQP 3070 EGA988 LPEGCHWAIWGRECLLPP 3071 EGA989 LPEGCWWANWGRECLLLP 3072 EGA991 IPEGCWWAQWGRECILAA 3073 EGA992 ISDGCFWAVWGRECLLAA 3074 EGA993 GTDGCWWEVWGRECLLTP 3075 EGA994 LPEGCWWSPWGRECLLPP 3076 EGA995 LTEPCYWTTWGKECHMPR 3077 EGA996 DEAGCWWEIWGRECILSP 3078 EGA997 IHDHCCWTIWGYECQLCP 3079 EGA998 LHDGCWWEVWGRECFVPS 3080 EGA999 FPDVCYWTVWGRECLLLP 3081 EGA1001 HPDGCYWTVWGRECLLPP 3082 EGA1002 YSEVCWWEVWGRECFLRS 3083 EGA1003 LPEDCWWAQWGRECFLPA 3084 EGA1004 LNDPCYWTTWGKECLLPR 3085 EGA1005 YEEHCGQFPTDWICTLIS 3086 EGA1006 YEEHCWLFPSDWICTLMT 3087 EGA1007 YEEHCWLFPTDWICTLVT 3088 EGA1008 YAEHCWLFPSDWICTLLT 3089 EGA1009 YAEHCCRFPSDWICTLMSGQA AAGGSQAQICRLLPSDRVCTL MP 3090 EGA1010 PESWCHTLYWNLQHCLSQE 3091 EGA1011 LPEGCYWATWGKECLLQP

TABLE 18 ErbB2 binding MRDs SEQ ID: Ref. No. Peptide Sequence 3113 EGB98 LCFAHQQAMFCN 3114 EGB104 TCHPSWGGKTCT 3115 EGB106 ICVAMTVTCS 3116 EGB117 FCIHFPHSWVCH 3117 EGB122 WCHLFPYSQWCH 3118 EGB123 ICKYFPFSHFCW 3119 EGB125 WDHWHIWHFG 3120 EGB127 LCETQPVSFSCSGQACPGGGGG SLCQFWPHIDFCV 3121 EGB128 WCHFQIWHHPCY 3122 EGB129 FCQFFPWTMVCQ 3123 EGB130 MPWEYLHQHQ 3124 EGB131 HCNWAPWWGYCMGQAGQGR CTAGGGSIICYWPPPNTRCW 3125 EGB132 PCVFVIEGCF 3126 EGB133 ICSRWPYWPHCWGQARQAAGG GSNCSHEPGSVVCL 3127 EGB134 WCRYYSVVCS 3128 EGB137 ICWIGTFGCA 3129 EGB221 PPYRCIPSESWICSFILGQA 3130 EGB222 QGGGCRGPHWYQMFYCVSPS 3131 EGB224 LVSFCPWGPDWYCSHYH 3132 EGB225 PPRWWCEHYGVQDLMCPGLF 3133 EGB227 SVFDCKYAPMWYCKHYL 3134 EGB249 GSLGCVLPMVFCRSYQ 3135 EGB263 YGKVLCSKWQCCKLLR 3136 EGB264 FCHLCQMFPAWCFLQT 3137 EGB289 RWVHCKQHGHYFSQRCIETW 3138 EGB310 PRRWCHMDFHHSPMCHWHL 3139 EGB315 HTRKCHKFMFQHKHCIEVL 3140 EGB334 MVVPCFTSQRQMAPACIAPRGQ AGQGGPGCGRWLPRQRCFIKH GHMSESCMVSA 3141 EGB335 YGKKCDKHDHSKPGVCIKKY 3142 EGB339 LLFDCWRIPEWHACGKDY 3143 EGB344 WNRICMYQWANHFTVCAPGP 3144 EGB350 ERALCHKPWSHIMPWCPQKL 3145 EGB354 WKPWCKLYWRADPACLQIK 3146 EGB356 WSGWCMYTWGWAPCRSVD 3147 EGB358 WSGWCVKQNAWYFCTGKI 3148 EGB364 VAQSSAFCEEFPVHWYCSFYVG 3149 EGB366 VAQSTPSCGMPKWYCTHYFG 3150 EGB367 VAQSSEHCYFGWNSYHCWSQAG 3151 EGB373 VAQREKHCVPEWYCKHYM 3152 EGB375 WSGWCWSDESKYWYSCHSRA 3153 EGB380 WSGLCLWDMFWQQCHGSS 3154 EGB383 SVLGCDDVGFYQCLEFLGQA 3155 EGB384 WSGWCQFKDGQWAKCTGRV 3156 EGB385 WSGYCFDSTHEGWYQCGAVS 3157 EGB386 WSGWCFHETHWQHCSAGG 3158 EGB387 WTGWCLVENQWTYCQNGD 3159 EGB388 WSGWCFEEAGWGHCMGLP 3160 EGB389 WSGYCFDRGYWQQCSSAW 3161 EGB391 WSGYCYVFDHWESCRGRE 3162 EGB392 WSGWCFKEAGWGHCMGLP 3163 EGB393 WSGCCHWEAAWNNCGGWT 3164 EGB394 WSGWCMISNQWRHCGSPD 3165 EGB395 WTGWCEGHGQWGWCHSSL 3166 EGB396 WVVSCHTSTQVLMCVCLL 3167 EGB398 WSGYCNSNGAWMMCGGGT 3168 EGB399 WSGWCFEQAGWGHCLGLP 3169 EGB400 WSGWCQSIIGEWMRCRSWT 3170 EGB401 WSGWCEEQFGWSQCRRSP 3171 EGB403 WSGWCFQETHWQHCMGLR 3172 EGB404 WTGFCFDENHWYICGTGR 3173 EGB406 WRGWCQIAGEWQHCSNYS 3174 EGB407 WTGYCVDQERHQWHQCFGRS 3175 EGB408 WQGYCYQEVYALWDHCSGPL 3176 EGB409 WSGWCFAMEHQKWKQCAGSS 3177 EGB410 GSMLCLWFNDNPQCFEVC 3178 EGB411 WSGWCETPHQWHDCKGTI 3179 EGB412 MIEFCWYYMALPECWIPT 3180 EGB413 IIEFCWYLQQYDECWVPK 3181 EGB414 WSGYCYEGVTWRSCWGDM 3182 EGB416 WSGWCFQFDNWDHCTGSV 3183 EGB419 SFTLTCGAHQCLCHVL 3184 EGB420 WSGWCQHMQVPIWHACSGGL 3185 EGB421 WSGLCLTEEVWHVCQGTI 3186 EGB423 WTGFCQSSKTQKWQHCRGGG 3187 EGB425 WSGWCEEELQWSFCSGHL 3188 EGB426 WSGYCVDFEFPAWGYCHGNI 3189 EGB427 WSGWCYSQALMSWTYCHEFK 3190 EGB428 WSGWCFYQVEYIWRSCEPAS 3191 EGB429 NRSGCIEIHYEDCLLIV 3192 EGB430 WTGWCYMEHFWDICHGPA 3193 EGB431 WTGYCLSSERHKWHHCFGRS 3194 EGB432 WSGWCFQFDNWDRCPGSV 3195 EGB433 WSGYCQMKDHWAHCGHSE 3196 EGB435 WSGWCLFADGWGHCYGVI 3197 EGB436 WSGWCFIKTHWGHCYGQI 3198 EGB437 WSGWCFYVDHWDYCGGLI 3199 EGB438 WSGYCEGTDHWYHCGGSM 3200 EGB439 WSGWCFYGDRWGHCSGLP 3201 EGB440 WSGWCFYGSYWGHCKGIL 3202 EGB441 WSGYCEFEDHWDYCGTPK 3203 EGB442 WSGWCLMETGWGHCKGLP 3204 EGB444 WSGWCFSADGWGIICRGVI 3205 EGB445 WSGYCDMGDHWSHCGHSG 3206 EGB446 WSGWCFYGSWWDYCSGGH 3207 EGB447 WSGYCEFEDHWDFCGTSR 3208 EGB448 WSGYCEYQDRWAHCGGTPGQ AGQGGGSWSGYCRTEGHWAH CGGSV 3209 EGB450 WSGWCLGEAGWGHCRGLL 3210 EGB451 WSGWCLYESGWGHCYGWI 3211 EGB452 WSGYCEYKYKWDHCGTSV 3212 EGB453 WSGYCEFKDHWAHCGGSL 3213 EGB455 WSGWCLGETGWGHCSGMP 3214 EGB456 WSGYCEFKDHWAHCGGSV 3215 EGB458 WSGYCEFQDHWGYCGNTE 3216 EGB459 WSGYCEFKDYWAHCSGSM 3217 EGB460 WSGYCEYSDHWAHCGGSL 3218 EGB462 WSGWCLYDAGWGYCSGLQ 3219 EGB463 WSGYCEMQDIIWDYCSGSI 3220 EGB464 WSGWCFTEGVWGHCKGLG 3221 EGB465 WSGYCEFKDHWDFCGHSA 3222 EGB469 WSGWCFLDSGWGHCQGLI 3223 EGB470 WSGWCFNSNGWYQCGGLI 3224 EGB471 WSGWCFFGASWGHCSGFP 3225 EGB472 WSGWCFFGAGWDYCHGQP 3226 EGB473 WSGYCEFKDHWDYCGGSE 3227 EGB474 WSGWCFYGTGWGYCYGLI 3228 EGB476 WSGYCEYKHHWAHCGTRH 3229 EGB477 WSGYCLMKDHWAHCGHSE 3230 EGB478 WSGWCMGMSGWDHCRGLI 3231 EGB479 WSGYCEINGKWDYCSHSH 3232 EGB480 WSGWCFFGAGWGHCFGNP 3233 EGB481 WSGWCLQGAGWGHCSGRI 3234 EGB482 WSGWCFNGVGWGHCFGLI 3235 EGB483 WSGWCFYEAFWGIICTGPT 3236 EGB484 WSGWCFDGDGWGYCTGVS 3237 EGB485 WSGWCLRSNGWDYCHGPT 3238 EGB486 WSGWCLSGAGWGYCSGLP 3239 EGB487 WSGWCFFGSAWGHCHGFP 3240 EGB488 WSGWCFYSTIIWAQCKGGII 3241 EGB489 WSGWCETDRGWSFCSSPL 3242 EGB490 WSGWCESELQWGFCSGLL 3243 EGB491 WSGWCEGETQWYFCSGHL 3244 EGB492 WSGWCEYELQWSFCSGQL 3245 EGB493 WSGWCEGEREWPFCSGHI 3246 EGB494 WSGWCEYEHHWDFCSGHL 3247 EGB495 LPPPCYWTTWGKECQMFD 3248 EGB496 WSGWCFSSSGWGFCGGLN 3249 EGB497 WSGWCFSPAGWGIICYGLS 3250 EGB498 WSGWCLDRAGWGHCHGLI 3251 EGB499 WSGWCLTKSGWGHCLGPT 3252 EGB500 WSGWCLYEFGWGYCSGLI 3253 EGB501 WSGWCEYELHWSYCWGPL 3254 EGB502 WSGWCEGELYWSYCSGYL 3255 EGB503 WSGWCEYELSWSFCSGSL 3256 EGB504 WSGWCEYEGFWSFCSGNL 3257 EGB505 WSGWCEGVLQWYFCSGHI 3258 EGB506 WSGWCEYDLHWSFCSGHL 3259 EGB507 WSGWCLGKAGWGHCGGLI 3260 EGB508 WSGWCFFGSIIWGIICFGSP 3261 EGB509 WSGWCLLDAEWGHCSGLI 3262 EGB510 WSGWCLSNDGWGHCWGLM 3263 EGB511 WSGWCLSGTGWGHCYGLH 3264 EGB513 WSGWCLVGTQWSFCSGLI 3265 EGB514 WSGWCEKEFLWTFCSGTI 3266 EGB515 WSGWCESDTHWSFCSGQL 3267 EGB516 WSGWCEGVSEWSFCSGLI 3268 EGB518 WSGWCLGLDGWDHCYGRP 3269 EGB519 WSGWCFFGDRWGHCHGQP 3270 EGB520 WSGWCFVGASWGHCYGMI 3271 EGB521 WSGWCLSDAGWGACGGAS 3272 EGB522 WSGWCLEWNGWGHCSGIP 3273 EGB523 WSGWCEGELQWFFCSGTI 3274 EGB524 WSGWCEGEHHWYFCRGNF 3275 EGB526 WSGYCEGEQQWFYCSGQV 3276 EGB527 WSGWCENKVGWSFCSGHI 3277 EGB528 WSGWCEGERTWHFCSGPL 3278 EGB530 WSGWCCFGTSWGYCHGPP 3279 EGB531 WSGWCLLDAGWGHCKGML 3280 EGB532 WSGWCFYGHGWGHCHGTP 3281 EGB533 WSGWCFDYTRWGIICSGMP 3282 EGB534 WSGWCLFSAGWGHCSGLI 3283 EGB535 WSGWCEGELQWSFCWGHI 3284 EGB537 WSGWCEEEHQWSFCSGYI 3285 EGB538 WSGWCEGEREWYFCSGFF 3286 EGB539 WSGWCEGKLKWNYCSGYL 3287 EGB540 WSGWCEQDQKWNYCSGYI 3288 EGB542 WSGWCFYEVRWGHCHGSI 3289 EGB543 WSGYCLSESGWGHCGGLL 3290 EGB544 WSGWCLSYSGWGHCSGSP 3291 EGB545 WSGWCFYGDYWGHCSGQP 3292 EGB546 WSGWCFKITGWGHCSGLI 3293 EGB547 WSGWCENELQWSFCSGHI 3294 EGB548 WSGWCEGERQWSFCSGSI 3295 EGB549 WSGWCEYEFKWSFCSGIII 3296 EGB550 WSGWCEHEMYWSYCSGHK 3297 EGB551 WSGWCEGELRWFFCSGTL 3298 EGB553 WSGWCFYGTHWGHCGGLP 3299 EGB554 WSGWCFDGGRWGYCSGPL 3300 EGB555 WSGWCLKGDGWGHCWGLI 3301 EGB556 WSGWCLRVSGWDHCSGHP 3302 EGB557 TDSWCHTLYWALQHCVAQE 3303 EGB558 WSGWCLMGDSWGHCSGLI 3304 EGB559 WSGWCEGEVGWFFCSGYL 3305 EGB560 WSGWCEYELQWSFCFGPS 3306 EGB561 PDSSCIITLYWQLIIIICARRE 3307 EGB562 WSGWCEGDLQWYYCSGHI 3308 EGB563 WSGWCEEALRWSYCSGHI 3309 EGB565 WSGWCLGDAGWGHCGGLP 3310 EGB566 WSGWCFFDTHWGHCSGLL 3311 EGB567 WSGWCFNHGAWGHCRGLI 3312 EGB568 WSGWCYEHTGWRHCHGLI 3313 EGB569 WSGWCLTREGWGHCKGLI 3314 EGB570 WSGWCLSGVGWFHCKGMI 3315 EGB571 WSGWCEGESQWFFCSGLI 3316 EGB572 WSGWCEYESHWSFCSGHI 3317 EGB573 WSGWCEGERKWSYCSGQI 3318 EGB574 WSGWCEGELHWYSCSGHM 3319 EGB575 PDSWCHTLYWDLLYCVNQE 3320 EGB577 WSGWCETPCGWESCHGTI 3321 EGB579 WSGWCESTDSWHFCRGNI 3322 EGB580 WSGWCETPSGWKACRGNI 3323 EGB581 WSGWCETGVGWHECHGTI 3324 EGB582 WSGWCEAERQWTFCSGHI 3325 EGB583 WSGWCENSEGWDFCNGTI 3326 EGB584 WSGWCETQKGWHSCHGSI 3327 EGB585 WSGWCLTGTDWIIFCRGFI 3328 EGB586 WSGWCETYKGWHACHGKI 3329 EGB587 WSGWCETNAGWIQCYGSI 3330 EGB588 WSGWCQTGANWHYCNGTI 3331 EGB589 WSGWCLADGMWGHCNGTT 3332 EGB590 WSGWCEGRDAWYQCKGTI 3333 EGB591 WSGYCLTDGTWHHCKGTI 3334 EGB592 WSGWCKTGKKWHHCGGTI 3335 EGB593 WSGWCETSSGWHQCSGII 3336 EGB595 WSGWCEEKGQWVSCGGSL 3337 EGB596 WSGWCEGELQWYFCSGHL 3338 EGB597 WSGWCEGEHKWSFCSGHL 3339 EGB598 WSGWCESDKGWYQCRGTI 3340 EGB599 YEEHCRLFPADWTCSLLQ 3341 EGB600 WSGWCETPGIIWYQCRGSI 3342 EGB601 ADSWCHTLYWNLRHCEIQE 3343 EGB602 WSGWCNSGGGWHQCRGTI 3344 EGB603 WSGWCLTDNKWHSCIGTI 3345 EGB604 WSGWCETDRGWHDCHGTI 3346 EGB605 WSGWCETKDHWHACNGII 3347 EGB606 WSGWCEGELQWYFCSGKI 3348 EGB607 PESWCHTLYWNLQHCLSQE 3349 EGB609 WSGWCETSGGWHYCKGTI 3350 EGB610 WSGWCETDQGWHHCWGTI 3351 EGB611 WSGWCETKFGWNYCKGSI 3352 EGB612 WSGWCFATSGWIISCRGTA 3353 EGB613 WSGWCETGNGWDRCWGNI 3354 EGB614 WSGWCEGQLQWFFCSGQI 3355 EGB615 WSGWCERSWGWEQCRGTI 3356 EGB616 WSGWCEYEEKWAFCSGYI 3357 EGB617 WSGYCEGELQWYFCTGHL 3358 EGB618 WSGWCETSVGWHKCSGTI 3359 EGB619 WSGWCRIETGWDFCSGTI 3360 EGB620 WSGWCETSKGWHFCSGTI 3361 EGB621 WSGWCESSHGWHGCKGII 3362 EGB622 WSGWCEYNGYWDYCTGTI 3363 EGB623 WSGWCETGKYWHYCSGTI 3364 EGB624 WSGWCEGKHQWYFCSGNL 3365 EGB625 WSGWCETELGWHQCRGTI 3366 EGB626 WSGWCEGQTKWYFCSGQI 3367 EGB627 WSGWCLFDSKWDYCQGNI 3368 EGB628 ANSWCHTLYWDLMYCVWQE 3369 EGB629 WSGWCQTGMRWHQCYGTI 3370 EGB630 WSGWCESNGTWHGCKGTI 3371 EGB631 WSGWCEGKHGWYSCRGTI 3372 EGB632 WSGWCETLSGWHQCHGVI 3373 EGB633 WSGWCEGKEKWIIIICNGSI 3374 EGB634 WSGWCETSEGWHSCYGSI 3375 EGB635 WSGWCLNELGWQQCFGTI 3376 EGB636 WSGWCERVSGWNYCHGTI 3377 EGB638 WSGWCYAPTGWHHCVGTT 3378 EGB639 WSGWCETGTGWYHCGGTI 3379 EGB640 PDSWCHTLYWHLQHCLSQE 3380 EGB641 WSGWCEYELRWDFCSGHI 3381 EGB642 WSGWCETVNGWHQCTGTI 3382 EGB643 WSGWCLTKNSWDFCSGTI 3383 EGB644 WSGWCLTNGVWHGCRGII 3384 EGB645 WSGFCLTEESWHHCTGPI 3385 EGB646 WSGWCEAIDMWYQCHGPI 3386 EGB647 WSGWCFTGDLWHACTGII 3387 EGB648 WSGWCEGELQWYYCSGKI 3388 EGB649 WSGWCEGEFQWYFCSGQF 3389 EGB650 WSGWCETNGKWDFCQGTI 3390 EGB651 WSGWCETPVGWEQCTGTI 3391 EGB652 WSGWCEGKLHWYFCSGHM 3392 EGB653 WSGWCEGALGWSSCSGHG 3393 EGB654 YEEHCALFPTDWICTLH 3394 EGB655 WSGWCEGIGAWHFCKGII 3395 EGB656 WSGWCESNDGWYQCKGDI 3396 EGB657 WSGWCETLNGWYGCHGTM 3397 EGB658 WSGWCENNDGWHHCHGSI 3398 EGB659 WSGYCETGRGWIIMCQGTI 3399 EGB660 WSGWCETVTGWDYCNGKI 3400 EGB661 WSGWCETDEGWRQCSGTI 3401 EGB662 WSGWCMTLDKWHQCYGTI 3402 EGB663 WSGWCETNNGWYACWGMI 3403 EGB664 WSGWCFNEAGWGHCYGLP 3404 EGB665 WSGWCETDRGWHECHGTI 3405 EGB666 WSGWCETKFGWSYCKGTI 3406 EGB668 WSGWCEGEDGWHSCRGTI 3407 EGB669 WSGWCETSKGWHFCSGII 3408 EGB670 PKYSCVYSIMKEKWHCRAQT 3409 EGB673 FKPHCVYKHQTWVCGPPA 3410 EGB675 THMHCYQHKHHYHCRTSL 3411 EGB686 GPELCHHMAGHEWCLRYY 3412 EGB692 HRSKCFMMYWDENCLQPW 3413 EGB693 LGGRCMLFALKQCSMGH 3414 EGB696 WSRLCTMRLPSTCGVWE 3415 EGB697 RSWHCSHQMGSKPHCVLSP 3416 EGB698 TWFRCTPHKHHYHCKHNV 3417 EGB705 GIWICRHDSPYMKPHCIFTS 3418 EGB707 TWSKCFMVPLGDYCTRAL 3419 EGB708 SVWKCVKIIPFKIKPLCKLES 3420 EGB710 RTWFCEKMHGQKFHCVSWF 3421 EGB712 ADSWCHTLFWQLQHCVWQE 3422 EGB714 KWRCCYGQFQPACNAVS 3423 EGB716 PRYKCVFNWEVQKWQCSVWS 3424 EGB722 PNRYCYTYPHSVKCAWMY 3425 EGB723 DPSRCSKAYQMLYQCRTLI 3426 EGB729 STDLCWPHKKHWHCPPAN 3427 EGB730 SAWYCTKHMPQSKYHCISMR 3428 EGB732 KWWKCGVIFEICQIVP 3429 EGB738 AQLKCFEKLYMCRHHL 3430 EGB739 VDRVCQFFMQKCWSSP 3431 EGB740 KWTRCIPRMETHTCDCLL 3432 EGB746 KLPQCLWEWDNRDCRWLL 3433 EGB748 LIIRFCDIIFAIQCRFCN 3434 EGB758 LICKQHKNHWHCRNYY 3435 EGB763 KPWKCFYSYKIQHWKCHGVS 3436 EGB770 TDSWCHTLYWQLHHCAILE 3437 EGB771 KWRCCYGQFQQACNAVS 3438 EGB778 WTHACVVWNMKQMEACTSLT 3439 EGB779 LLNQCLQWAFHNCGCWN 3440 EGB780 WSGYCELKDRWAYCSGSI 3441 EGB781 HRAPCFSCTACRQPCFSLT 3442 EGB785 WSGYCEMKGYWAHCTGLI 3443 EGB786 WSGYCEFLDHWDHCSGGE 3444 EGB787 WSGYCEYKDIIWDIICGSSV 3445 EGB788 WSGYCEYKDHWDYCGSSV 3446 EGB789 WSGYCEYKDHWDFCGNVD 3447 EGB791 WSGYCEYRDHWAHCGGSL 3448 EGB792 WSGYCEFEGYWAHCGGSI 3449 EGB795 WSGYCEYIDHWGYCQGSI 3450 EGB796 WSGYCEMNDHWDYCYGSI 3451 EGB798 WSGYCEYKDHWGYCGGSI 3452 EGB799 WSGYCQLMDHWDYCHGQG 3453 EGB802 WSGYCEMTDHWAHCSGSH 3454 EGB803 WSGYCEFEDHWDYCHGSV 3455 EGB805 WSGYCEMKDHWAHCGGLI 3456 EGB806 WSGYCEFKDHWDYCHGSV 3457 EGB807 WSGYCEMGDHWDYCSGSV 3458 EGB808 WSGYCEFNGFWAHCGGFI 3459 EGB809 WSGYCLMKDHWGYCGGWI 3460 EGB810 WSGYCEFEDHWDYCGNSE 3461 EGB811 FQGHCALFPTDWICTLKS 3462 EGB813 WSGYCEFKTHWGHCGNPQ 3463 EGB814 WSGYCEFKDHWDHCHSSG 3464 EGB816 WSGYCEKEGYWAHCRGTI 3465 EGB817 WSGYCEMKGIIWAIICGGSP 3466 EGB818 WSGYCNMGDHWDHCSGSI 3467 EGB820 WSGYCEFKDHWAHCSGSL 3468 EGB822 WSGYCEMKGYWDFCGGLI 3469 EGB823 WSGYCEFQDYWDYCGGSI 3470 EGB824 WSGYCEMEHGWDYCSGTI 3471 EGB825 WSGYCEFKDHWDHCGDSE 3472 EGB826 WSGYCELEDHWAFCGGTI 3473 EGB827 WSGYCEFKGYWDFCYGSM 3474 EGB829 WSGYCEGQYFWYHCSGSG 3475 EGB830 WSGYCEFQDHWDHCFGSS 3476 EGB831 WSGYCEMNDHWDYCYGPK 3477 EGB832 WSGYCEFRDHWDYCGNSY 3478 EGB834 WSGYCEFKDHWDYCGNSD 3479 EGB835 WSGYCEFKDIIWDFCGGSY 3480 EGB836 WSGYCQTNDHWDYCSGTI 3481 EGB837 WSGYCESYDHWYHCGGTL 3482 EGB838 WSGYCEYEDHWDYCGTWV 3483 EGB839 WSGYCEMEDRWAYCSGSI 3484 EGB840 WSGYCEFKDHWDHCGGSL 3485 EGB845 WSGYCQMKDHWDYCSGSL 3486 EGB847 WSGYCEGKEHWYQCGGPL 3487 EGB848 WSGYCEFNDHWAHCHGSP 3488 EGB849 WSGYCEFKDRWAHCSGSI 3489 EGB850 WSGYCEFKGYWDHCGSSK 3490 EGB851 WSGYCEYKDRWAIICRGTM 3491 EGB852 WSGYCEFKDHWDYCGGSY 3492 EGB853 WSGYCEMKDHWAHCSGSI 3493 EGB854 WSGYCEYQDHWAHCGRSM 3494 EGB855 WSGYCEFSDHWDHCGNSK 3495 EGB857 WSGYCEMKDHWAHCGGSI

TABLE 19 ErbB3 binding MRDs SEQ ID: Ref. No. Peptide Sequence 3499 EGC1 VYQLCIKMWVCTWHG 3500 EGC2 DLVGCLLFSHVSCFSPL 3501 EGC3 PTLSACMEAKCVLIFL 3502 EGC5 LLWRCELMPPWPYMCSEVD 3503 EGC13 GTQECAQDDYFRYWCLRTW 3504 EGC14 LDLCMTIERPHRQCPCG 3505 EGC19 LWRWCVWARDCPQSS 3506 EGC20 IAREFCMNYQCLRLLR 3507 EGC21 SYWVCLTYFICWPSW 3508 EGC39 VQQFVHWMESWNPACFWRM 3509 EGC40 LLNQCLQWAFHNCGCWN 3510 EGC44 LSWWCRMGWFQEYGECNFLF 3511 EGC48 WTHACVVWNMKQMEACTSLT 3512 EGC54 KVMFCWVMWKQCSSPP 3513 EGC93 SLVSCPVHNPIWWSHCMEIM 3514 EGC94 GHRLCIWMQNFVDGHCAGLV 3515 EGC99 CHVSCPFYNPNWWSHCMELM 3516 EGC100 FLEMCMDWNPKTWCYCMEWM 3517 EGC101 SFVMCPVYNPKQWSVCMEIL 3518 EGC102 LFLMCLVYPSEKWHVCVETL 3519 EGC104 SLVKCPFFNPIWWSHCMELM 3520 EGC106 WSHACIVYNDKIMGACTCLT 3521 EGC115 DQVRCPFQNPVWWSHCMYLL 3522 EGC117 PLERCPFQNPLWFSHCMEML 3523 EGC120 KFFMCQVYPSNRWAVCVETL 3524 EGC121 TFYVCQIYPSGKWHVCVGTC 3525 EGC122 RFFMCQVYPSYKWHVCVESW 3526 EGC123 KFLMCRLYPSGKWHVCVETL 3527 EGC125 LEVYCPFENPIWWARCMELM 3528 EGC127 SRVRCPFQNPIWEAHCMKMT 3529 EGC128 SQVRCPFQNPIWRNHCMELI 3530 EGC129 PKVRCPFQNPIWWSHCMEML 3531 EGC130 NFVMCQVYPSYTWHVCVGTL 3532 EGC132 KFMICRLYPSGNLHICVETS 3533 EGC133 TFMMCQVYPSLKWHVCIETG 3534 EGC134 RFVMCQVYPSLNWHVCVQTL 3535 EGC135 SPVRCPFQNPIWYAHCMKIT 3536 EGC136 SKVYCPFQNPIWFAKCIELM 3537 EGC138 PQVRCPFQNPIWYSHCMKLM 3538 EGC139 TQVRCPFQNPIWWSHCMEML 3539 EGC140 KLMLCQVYPSLKWHVCVEAS 3540 EGC141 NFLMCQVYPSYKWHVCVEQL 3541 EGC142 RFLVCQVYPSLKWHVCVEKS 3542 EGC143 TFFICQVYPSGKWHVCVDTV 3543 EGC144 GFIMCQVYPSENWHVCLDRG 3544 EGC145 KFIMCQVYPSERWHVCLETM 3545 EGC150 SDVYCPFSNPAWWVHCMEML 3546 EGC151 TQVYCPFANPVWWNIICMLLS 3547 EGC152 KFFMCQIYPSLKWHVCVEKL 3548 EGC153 KFVMCQVYPSEKWHVCIAPL 3549 EGC154 QFFMCQVYPSLRWSVCQVTL 3550 EGC155 GFVMCQVYPSQKWHVCLDTF 3551 EGC156 GFHMCQVYPSGKWLVCLHPR 3552 EGC157 EFYMCQVYPSELWHVCVDKL 3553 EGC158 FKERCPFQNPIWYSRCMELI 3554 EGC159 SSVRCPFQNPIWWSHCMELL 3555 EGC160 SEIRCPFKNPIWYAHCMKLI 3556 EGC161 SQVRCPFQNPIWYTHCMELL 3557 EGC163 SQVYCPFIINPIWLAIICMELM 3558 EGC164 KFILCQVYPSGKWHICTETL 3559 EGC165 NLLLCKVYPSGKWHVCFAPL 3560 EGC166 KFQLCQVYPSERWHICVVTL 3561 EGC167 NFYMCKVYPSELWHVCVETL 3562 EGC168 RFVMCQVYPSAKWHVCVETL 3563 EGC169 TNIRCPFQNPIWYSHCIALT 3564 EGC171 SPVRCPFQNPIWYEHCMALI 3565 EGC172 SAVRCPFFTNPIWYSHCMKLL 3566 EGC174 AQVRCPFQNPVWYSHCMDLI 3567 EGC175 YFRMCQVYPSLKWHICVETM 3568 EGC176 GFFMCQVYPSEKWHVCLETL 3569 EGC177 GFIMCQVYPSLKWHVCVETS 3570 EGC178 KFFMCQIYPSEKWHVCVKLW 3571 EGC181 AASLCPFQNPIWLAHCMEMM 3572 EGC182 SKVYCPFQNPIWWSHCMELI 3573 EGC183 SQAYCPFQNPIWWNHCMELI 3574 EGC185 HLFLCQVYPSEKWHVCVETL 3575 EGC186 KFRMCQVYPSGKWHVCAETF 3576 EGC187 KFFLCQVYPSGKYHICVSTW 3577 EGC189 KFYMCQVYPSVRWHVCVDTS 3578 EGC191 TREICPFQNPIWRSIICLKLV 3579 EGC192 PMVRCPFKNPVWYSHCLELL 3580 EGC196 TFFMCQVYPSGKWHVCVGGL 3581 EGC197 KFFLCQVYPSEKWHVCIERL 3582 EGC198 RYQMCQVYPSGKWHVCMETT 3583 EGC199 RFQMCKVYPSERWHVCVEIL 3584 EGC222 WCTECMRRFPGHYCVAFC 3585 EGC223 RAASCLDVFHWCHSWC 3586 EGC224 CLSRCVDWVFSNCTSGR 3587 EGC225 FLMLCHVSKYTIKCFSPL 3588 EGC226 WALACSWTPRWHQCIFSL 3589 EGC227 LERLCMEIFYVHCSGSI 3590 EGC228 SWRMCIEMFLTHDECVRFV 3591 EGC229 SSVVCVRGIHAWTCHFFV 3592 EGC232 LTSSCVYVYNWCAVWA 3593 EGC233 YRHLCVWLMEQCETAR 3594 EGC234 RAASCLDVFHCRRSWS 3595 EGC236 HVSICWGYTYCWQIF 3596 EGC237 RGASCLDLFHWCHSWG 3597 EGC238 IYILCTNFKPVFWCVFST 3598 EGC239 LWIRCSLLQYSYYCSWVW 3599 EGC242 SYLFCYTLYIDYNCWLWS 3600 EGC243 PGYRCVFQVNYQCGAAF 3601 EGC244 TIIRCFWTFHMDFVCVTGV 3602 EGC245 WLNCGCVVITCIVEPR 3603 EGC246 WGWRCYQSWSYQDCQYWY 3604 EGC248 FLMLCRVSKYTIKCFSPL 3605 EGC250 RGRDCLRSFPGHYCVTFR 3606 EGC251 LNYICWRWAHQVPWQCSLFQ 3607 EGC252 ASYSCWWGPMYMECWTKV 3608 EGC253 KTGSCQAQKVFSWCAQWC 3609 EGC254 KFFMCQVYPSLKWHVCMETL 3610 EGC255 KFMMCQVYPSLKWHVCVETL 3611 EGC256 VEHLCSKIPFWHVQCLARF 3612 EGC259 KFLMCQVYPSLKWHVCMETL 3613 EGC263 QFLMCQVYPSLKWHVCVETL 3614 EGC264 QFMLCQVYPSKKWHVCVVPV 3615 EGC265 GSCALKNRSKCARRG 3616 EGC266 KFQMCQVYPSGNWHVCVETM 3617 EGC267 RERTCVYFHKILHDFCPSRT 3618 EGC268 FKFTCYWAPYHTFCNTGQ 3619 EGC269 KFFMCQVYPSLKWHVWMETL 3620 EGC270 KFQCARFTLPETGMFVSKL 3621 EGC272 WYEQCMKRHHPQAFCLRFN 3622 EGC273 KFLMCQVYPSKKWHVCIETL 3623 EGC274 QFMLCQVYPSKKWHVCVVTV 3624 EGC276 CLGSCMLKGFERWCIIIGII 3625 EGC277 KFFMCVDYPSLKWDVCMETL 3626 EGC278 RFFMCVDYPSIKWDVCPETI 3627 EGC280 PRWTCEWYQARQSCSLGQ 3628 EGC281 KFHMCQVYPSRNWHVWVETL 3629 EGC283 VEHLCSEIPFWNVQCLARF 3630 EGC284 KLFLCQVYPSERWHVCVESL 3631 EGC285 HLIMCQVYPSGKWHVCLATT 3632 EGC286 AVELCFHHTQEWTYCLWRF 3633 EGC287 GEQLCSKIPSSDALWWVRLL 3634 EGC288 DALRCFEWVKRNCLLSS 3635 EGC289 KFMMCQVYPSQKWHVCVETF 3636 EGC291 VEHLCSKIPFWHVQCLARI 3637 EGC337 RYFYMCQVYPSERWQVCVGTS 3638 EGC453 SNLYLCQVYPSGKYIIICIEAL 3639 EGC465 YSEPYCPFLNPTWKNECIARS 3640 EGC466 TSEPYCPFLNPIWKAECISRT 3641 EGC467 SYEPYCPFLNPTWKEQCIIRS 3642 EGC468 LNEPYCPFMNPNWKAECLARM 3643 EGC469 SKGPYCPFLNPIWKDECIARI 3644 EGC470 NSEPYCPFINPIWKAECIART 3645 EGC471 QSKPYCPFLNPTWKAQCIARS 3646 EGC472 TSTPYCPFLNPIWKAECLSRI 3647 EGC474 ISEPYCPFMNPTWKEQCLART 3648 EGC476 VNDPYCPFLNPTWKAECIART 3649 EGC488 SIEPYCPFLNPTWKAECLVRS 3650 EGC489 VSEPYCPFLNPVWKDQCLSRI 3651 EGC490 LPPPCYWTTWGKECQMFD 3652 EGC491 KTEPYCPFLNPTWKAECIART 3653 EGC492 TSDPYCPFVNPIWYNECIART 3654 EGC494 TGSPYCPFLNPTWKAHCIARL 3655 EGC500 ASEPYCPFLNPTWKDQCMARL 3656 EGC501 RSEPYCPFLNPTWKAQCIYRT 3657 EGC502 SIDPYCPFLNPTWLKECIARS 3658 EGC508 IGEPYCPFANPTWKQECIART 3659 EGC511 WSEPYCPFLNPAWKDQCLART 3660 EGC512 MREPYCPFLNPTWKDQCIAIR 3661 EGC514 YREPYCPFLNPIWKEQCIARS 3662 EGC515 ISAPYCPFLNPIWKDECIART 3663 EGC516 LREPYCPFLNPIWNVECLARS 3664 EGC517 SSEPYCPFHNPTWKDQCLARS 3665 EGC518 DSEPYCPFLNPTWKDQCLARF 3666 EGC519 NSEPYCPFLNPSWKASCIARM 3667 EGC522 TSEPYCPFLNPIWKDQCIART 3668 EGC523 RSEPYCPFLNPIWKNECIARS 3669 EGC524 FSEPYCPFIPTWKAQCLARI 3670 EGC525 LNEPYCPFLNPIWKDECLART 3671 EGC526 SREIYCPFLNPIWLAECIARA 3672 EGC528 LTEPYCPFLNPTWKDQCLARR 3673 EGC529 SKDPYCPFVNPIWKHECIVRS 3674 EGC532 HSEPYCPFLNPIWKEQCMVRS 3675 EGC533 SSEPYCPFLNPTWKIIECLLRT 3676 EGC534 ESEPYCPFTNPTWKAQCIARS 3677 EGC535 YNEPYCPFINPIWKAECIART 3678 EGC536 ISEPYCPFQNPTWKFQCLIRS 3679 EGC538 RSEPYCPFLNPTWKEQCIARQ 3680 EGC539 GRETYCPFLNPIWKAQCLART 3681 EGC540 KSEPYCPFLNPIWKDQCIVRS 3682 EGC541 VIEPYCPFLNPTWKDHCIART 3683 EGC542 MSDPYCPFQNPIWKAACIART 3684 EGC544 ESEPYCPFVNPIWKAECIARH 3685 EGC546 YNEPYCPFVNPTWKAACVART 3686 EGC548 YSKPYCPFINPIWKEQCIARH 3687 EGC554 LIEPYCPFLNPTWYDQCIARS 3688 EGC557 REVPYCPFLNPIWKEQCIIRT 3689 EGC561 SLQAYCPFVNPTWKNECLARI 3690 EGC563 LSEAYCPFLNPTWKQHCIALT 3691 EGC564 MSEPYCPFLNPTWKEQCLARS 3692 EGC565 LNEPYCPFLNPIWKNQCIARL 3693 EGC566 PSKPYCPFLNPYWKAECMSRS 3694 EGC568 ISEPYCPFLNPTWKDACIARS 3695 EGC570 PDSWCHTLYWDLLYCVNQE 3696 EGC572 QGQPYCPFVNPIWYAECMTRT 3697 EGC578 RPEPYCPFKNPNWKEQCLARY 3698 EGC579 SSEPYCPFINPTWKQECIART 3699 EGC582 TTEPYCPFLNPIWKDQCIARW 3700 EGC583 VSEPYCPFLNPYWKDQCIARH 3701 EGC584 YNEPYCPFLNPTWKDQCLARI 3702 EGC585 RSEPYCPFYNPIWYDHCVALT 3703 EGC586 NPEPYCPFLNPIWKEQCILRS 3704 EGC589 LSGLYCPFLNPTWKDQCIARM 3705 EGC590 ISQPYCPFTNPTWLAECIARS 3706 EGC592 LNTPYCPFINPTWKDECLARS 3707 EGC594 YEEHCRLFPADWICSLLQ 3708 EGC595 NSEPYCPFLNPSWKAQCIARS 3709 EGC596 ADSWCHTLYWNLRHCEIQE 3710 EGC597 SSEPYCPFLNPTWKEQCIART 3711 EGC599 DTKPYCPFLNPIWKNQCIERT 3712 EGC600 TRDPYCPFLNPIWKDECFART 3713 EGC601 KEEAYCPFLNPLWKDECVART 3714 EGC602 PESWCHTLYWNLQHCLSQE 3715 EGC603 MSEPYCPFQNPTWKIIECIART 3716 EGC604 DSDPYCPFVNPIWKAQCIARS 3717 EGC605 YSEPYCPFVNPTWLDQCIART 3718 EGC606 YSEPYCPFLNPTWKAQCMAMI 3719 EGC608 LSEPYCPFLNPTWKAACLARI 3720 EGC609 IRKPYCPFLNPTWYNECIQRS 3721 EGC610 QGEPYCPFLNPAWKDACIERL 3722 EGC612 GSEPYCPFFNPIWKDQCITRF 3723 EGC613 RSEPYCPFLNPTWKEHCIART 3724 EGC614 TNEPYCPFLNPTWKQQCIART 3725 EGC615 TSEPYCPFENPIWKAQCISRS 3726 EGC616 KSEPYCPFLNPYWKDQCIARS 3727 EGC617 LREPYCPFLNPTWKAECIARI 3728 EGC619 TNDPYCPFLNPTWKAQCLDRY 3729 EGC620 KGQPYCPFLNPIWKEQCLART 3730 EGC621 SSEAYCPFLNPTWKQECIARL 3731 EGC623 ANSWCHTLYWDLMYCVWQE 3732 EGC624 TIEPYCPFLNPIWKDQCIARS 3733 EGC625 QHEPYCPFLNPVWKHECIART 3734 EGC626 IREPYCPFLNPTWKAQCIART 3735 EGC627 HSAPYCPFLNPIWKDECIARS 3736 EGC628 VSEPYCPFLNPTWKAHCIELS 3737 EGC629 SSDPYCPFLNPTWYDQCIERY 3738 EGC630 ISDPYCPFVNPYWKAECIART 3739 EGC631 NIDPYCPFLNPTWKAECIARI 3740 EGC632 PREPYCPFLNPLWKNQCIERT 3741 EGC634 IRVPYCPFQNPTWYDQCIART 3742 EGC635 PDSWCHTLYWHLQHCLSQE 3743 EGC636 TNDPYCPFLNPTWKAECIARH 3744 EGC637 VSEPYCPFLNPTWKAQCLNRI 3745 EGC638 PIDPYCPFLNPIWKEQCHARS 3746 EGC639 VREPYCPFLNPTWKNECIART 3747 EGC640 LIEPYCPFLNPIWKDQCLART 3748 EGC641 PKFFMCQVYPSGKWHVCTGTV 3749 EGC642 PKYYMCQIYPSLKWNVCLETL 3750 EGC644 LNFYICKLYPSANLHVCVDTL 3751 EGC646 RSEPYCPFLNPVWKDQCIARS 3752 EGC647 SSEPYCPFLNPIWKYHCIARS 3753 EGC648 PGSPYCPFLNPFWKEQCIVRT 3754 EGC649 YEEHCALFPTDWICTLH 3755 EGC651 FIEPYCPFINPTWKAECLART 3756 EGC652 DKFYLCQIYPSERWHVCVWSS 3757 EGC653 EKFKMCQIYPSKNWAVCVETF 3758 EGC654 HKFYMCQVYPSEKWHVCVETL 3759 EGC655 LSEPYCPFLNPSWKAECIARS 3760 EGC656 LSEPYCPFLNPIWKEQCIARS 3761 EGC657 LSEPYCPFLNPTWKDQCIARS 3762 EGC658 RSEPYCPFVNPIWKAECIVRS 3763 EGC659 QFEPYCPFLNPTWKEQCIARI 3764 EGC660 PIEPYCPFQNPTWKAQCIART 3765 EGC661 YRQPYCPFLNPTWKAQCMART 3766 EGC663 DNFYMCQVYPSLKWIIICVSIL 3767 EGC664 SMFFMCQVYPSEKWHVCVYPL 3768 EGC665 SKFFMCQVYPSERWHVCIEPL 3769 EGC666 LKFYMCQVYPSGNWNVCTATQ 3770 EGC667 NSEPYCPFHNPTWKAACMART 3771 EGC668 RSEPYCPFLNPIWKEQCIART 3772 EGC669 SREPYCPFLNPTWKEQCIARI 3773 EGC671 MREPYCPFLNPSWKEQCIARS 3774 EGC672 LREPYCPFLNPVWKNECIART 3775 EGC673 ITDTYCPFQNPIWKAECIART 3776 EGC674 FNFFMCQVYPSEKWHICVKSL 3777 EGC675 KKFYMCQIYPSEKWHVCVERI 3778 EGC676 TRYSMCQVYPSLRWIIVCVENL 3779 EGC678 LGEPYCPFLNPTWKAQCLYMS 3780 EGC679 SSEPYCPFLNPIWKNECLARQ 3781 EGC680 RSEPYCPFLNPIWKEHCINRS 3782 EGC681 HSQPYCPFQNPTWYEQCIART 3783 EGC682 SNEPYCPFLNPSWKDACLQRT 3784 EGC683 LSDVYCPFVNPTWKAECIARS 3785 EGC684 SDFFMCQVYPSQKWHVCVKSL 3786 EGC686 PDSWCHTLYWSLLHCARQE 3787 EGC687 LPFFMCQVYPSKSWHVCVETS 3788 EGC688 LSEPYCPFQNPTWKEQCVARS 3789 EGC689 ADSWCHTLFWNLKHCLIQE 3790 EGC690 GGDPYCPFLNPTWKAECIART 3791 EGC691 VREPYCPFLNPIWKDQCIART 3792 EGC692 ASEPYCPFLNPIWKDQCIART 3793 EGC693 SSEPYCPFQNPTWKDQCIARN 3794 EGC694 ADSWCHTLYWNLLYCANQE 3795 EGC696 SWFFMCQIYPSLKWHVCVETL 3796 EGC698 STFYMCKVYPSQRWHVCVDTL 3797 EGC700 SREPYCPFLNPTWYDECIART 3798 EGC701 QSEPYCPFLNPMWKEQCIVRI 3799 EGC702 TSEPYCPFDNPTWKARCLAML 3800 EGC703 ARQPYCPFLNPTWKNECMARS 3801 EGC704 PSEPYCPFLNPIWKAECIART 3802 EGC705 IAILTALSLTLSGRTSASSE 3803 EGC706 YSAPDCPFVNPIWKNACIARY 3804 EGC707 ADSWCHTLFWQLQHCVWQE 3805 EGC708 LNFSMCQVYPSYKWHVCVGTL 3806 EGC709 MEFFMCQVYPSEKWHVCVAKL 3807 EGC710 LRYYICQIYPSEKYHVCVDTL 3808 EGC711 VIEPYCPFLNPTWKAECLARS 3809 EGC712 STEPYCPFLNPIWKDACIARH 3810 EGC715 WRDPYCPFLNPIWKEQCIARY 3811 EGC716 RSEPYCPFLNPTWKDQCMART 3812 EGC717 VIDPYCPFLNPIWKEQCQVRT 3813 EGC718 SKFFMCQVYPSKKWHVCVEAL 3814 EGC719 IKFFMCQVYPSELWHVCVQKQ 3815 EGC720 TTFFMCQLYPSGNWHVCTETL 3816 EGC721 FKFFMCQTYPSEKWIIVCVEKL 3817 EGC722 SSEPYCPFLNPLWHEQCLARI 3818 EGC723 AREPYCPFLNPIWKAECVARY 3819 EGC724 PSEPYCPFVNPTWLDECIARS 3820 EGC725 ASEPYCPFLNPTWYEQCINRT 3821 EGC726 PISPYCPFLNPIWYEECIARS 3822 EGC727 VGEPYCPFMNPIWKEQCILRS 3823 EGC729 ADSWCHTLYWQLLHCVNQE 3824 EGC736 ADSWCHTLYWQLLHCVSQE 3825 EGC737 DQVRCPFQNPVWWSHCMYLL 3826 EGC745 DQVRCPFQNPVWWSHCMYLL 3827 EGC746 DQVRCPFQNPVWWSHCMYLL 3828 EGC752 YTKHCQRLPTYKICNLFM 3829 EGC754 DQVRCPFQNPVWWSHCMYLL 3830 EGC755 ADSWCHTLYWQLQHCLSQE 3831 EGC759 DQVRCPFQNPVWWSHCMYLL 3832 EGC763 DQVRCPFQNPVWWSIICMYLL 3833 EGC764 DQVRCPFQNPVWWSHCMYLL 3834 EGC765 TDSWCHTLYWQLHHCAILE 3835 EGC770 DQVRCPFQNPVWWSHCMYLL 3836 EGC771 YQVTCPFLNPPWWSHCMYLL 3837 EGC772 DQVRCPFQNPVWWSHCMYLL 3838 EGC776 HRAPCFSCTACRQPCFSLT 3839 EGC777 YQVTCPFFNPLWWSHCMYLL 3840 EGC783 SEPYCPFLNPTWKSQCIART 3841 EGC789 YEVQCPFQNPIWWKHCMDLI 3842 EGC791 ESCWTWDHWYQYWVPQRCDP 3843 EGC792 ANSWCHTLYWQLHHCEILE 3844 EGC793 QACNFTSLGWYQEFYCLR 3845 EGC794 WACRWEWHAAWQKWDEFCVE 3846 EGC795 AYCYRSIISRNSTCSAL 3847 EGC796 VYCFLNFKTMNYAGEFWCRH 3848 EGC797 SWCMWYYESTYSAPYYYCSV 3849 EGC798 SQCHWWHEPYGMFQTEICLW 3850 EGC799 GLHGCQYVQMFFHFCAPKT 3851 EGC800 WQHVCWQDWYSWVCTSGY 3852 EGC801 PWRGCWWDPWQPWDCVPFF 3853 EGC802 QERLCRQIWQIIYCVRYM 3854 EGC803 QPRECNYVPAWYCHYYS 3855 EGC804 VYQYHCDWYTCLFRHT 3856 EGC806 FQGHCALFPTDWICTLKS 3857 EGC807 QMCHMEWHNHVYWQVCHW 3858 EGC808 WMRQCHLETECQRAS 3859 EGC809 SAVACFHPPYFFICVASG 3860 EGC810 LRRRCHSRFMQCSNPG 3861 EGC811 GQTWCHWGMYCISTT 3862 EGC818 WEYQCEWGYSYKPQCWLSA 3863 EGC820 LPLTCWVLEWHEWLACVSYD 3864 EGC822 WSYSCVFLPWMYHMCYLET 3865 EGC823 WFYQCWQSPWHTECSWVN 3866 EGC824 RDQPCWYKIMTGCLCMP 3867 EGC825 LPPSWCVRGECSWLLL 3868 EGC826 DESDCKQLPICHGHCLVPL 3869 EGC827 YQVFCEWSYWEYHCISNQ 3870 EGC828 VQHHCLYRQHSYRECVRHY 3871 EGC829 YIDCVPVLPGFYLPQLG 3872 EGC831 WHQACEYQYYNWECSMGW 3873 EGC832 ETFSCYVFPFFYECYSTP 3874 EGC836 WTNLFCWGPLCKCLQQ 3875 EGC839 YQVMCLHQPFVSFCHYET 3876 EGC840 GTHWCRYYPSYAECYVWL 3877 EGC841 TWLCDCSYWGCWWELG 3878 EGC842 LWVGCYIPPWTEWCPIAW 3879 EGC844 VWLMCWHYIFCPSKL 3880 EGC845 PEYLCWVYPYAPQCWDLV 3881 EGC846 VEHLCSKIPFWMFSAWPG 3882 EGC849 ICEACPKIIVSEQCIRLR 3883 EGC851 WRVICMARPTGEQCEWVW 3884 EGC854 DWSKCIQFSLPHDHCLMFM 3885 EGC856 SRWECAWIVGYHHWDCRWLF 3886 EGC857 SMEYCFWYHYCQLGV 3887 EGC858 IFVMCEDSWPVPFCFSNR 3888 EGC859 VEWMCYAYPFDPECYYNW 3889 EGC861 DEHLCSISRSGMSFAWAG 3890 EGC862 ASEPYCPFLNPVWKEQCIARL 3891 EGC863 PREPYCPFHNPIWKNQCIART 3892 EGC865 ESEPYCPFLNPIWKAHCIART 3893 EGC866 DSEPYCPFLNPIWKEQCLARM 3894 EGC867 TSDPYCPFLNPTWKAECIART 3895 EGC868 WSEPYCPFTNPTWKAACIARS 3896 EGC869 ARDPYCPFQNPIWYNECIART 3897 EGC870 PREPYCPFHNPAWKAACVARA 3898 EGC871 TNESYCPFLNPTWKAECMARI 3899 EGC872 DREPYCPFLNPYWKDQCLART 3900 EGC873 ISDPYCPFVNPIWKEQCIARN 3901 EGC874 RNEPYCPFVNPIWKEQCIIRT 3902 EGC875 LTTPYCPFENPTWKAQCIART 3903 EGC876 SIEPYCPFLNPTWLEQCIARS 3904 EGC877 KSEPYCPFLNPTWHDQCIARN 3905 EGC878 IIGPYCPFLNPEWKDQCMART 3906 EGC879 YREPYCPFQNPYWKDQCIARY 3907 EGC880 AIEPYCPFLNPIWKDQCLARI 3908 EGC881 MSRPYCPFLNPIWKEQCIART 3909 EGC882 YNKPYCPFQNPTWKEACLARS 3910 EGC883 SSEPYCPFLNPTWKAECIARS 3911 EGC884 LSEPYCPFQNPIWKDKCIAMS 3912 EGC885 SYEPYCPFLNPTWKAECIARI 3913 EGC886 GSEPYCPFLNPTWKEQCIIRT 3914 EGC888 SSEPYCPFLNPTWKSQCIARS 3915 EGC889 RSEPYCPFLNPIWKDHCIART 3916 EGC890 RSEPYCPFLNPTWKAECIARS 3917 EGC891 SREPYCPFLNPIWKDKCISRT 3918 EGC892 TSDPYCPFLNPIWKEQCIVRT 3919 EGC893 EREPYCPFLNPTWKEQCLARH 3920 EGC894 LREPYCPFLNPTWKAECIARS 3921 EGC895 GSEPYCPFLNPTWKEQCLARL 3922 EGC896 MSEPYCPFLNPIWKEQCIARS 3923 EGC897 RGEPYCPFTNPIWKEQCINRS 3924 EGC898 DADPYCPFLNPTWKAECLARS 3925 EGC899 RGEPYCPFLNPAWKNECLART 3926 EGC900 IIEPYCPFVNPNWKAQCIARS 3927 EGC901 EDEPYCPFLNPIWKAQCLART 3928 EGC902 FTEPYCPFINPTWKAECIARS 3929 EGC903 TSEPYCPFLNPIWKEQCITRT 3930 EGC904 VEPYCPFLNPTWKDACLART 3931 EGC905 TSEPYCPFGNPIWKKECIART 3932 EGC906 TSEPYCPFLNPTWKDECIARM 3933 EGC907 SNEPYCPFLNPTWKAQCLARM 3934 EGC908 YREPYCPFLNPTWKEQCIARS 3935 EGC909 LNAPYCPFLNPNWYDQCIHRS 3936 EGC910 SSDPYCPFLNPTWKQQCIERS 3937 EGC911 ASEPYCPFLNPTWKEQCIART 3938 EGC912 LSEPYCPFQNPTWYDECISRS 3939 EGC913 SSEPYCPFLNPKWKHECIARS 3940 EGC914 GSEPYCPFLNPIWKAECIARS 3941 EGC915 RIEPYCPFVNPTWKDQCLART 3942 EGC916 SIEPYCPFLNPTWKEQCIARS 3943 EGC917 RTEPYCPFLNPTWKEQCIARS 3944 EGC918 PSEPYCPFLNPTWKDQCLQRS 3945 EGC919 YSEPYCPFLNPTWKAECIART 3946 EGC920 TSEPYCPFLNPTWKEHCMART 3947 EGC921 GSNPYCPFLNPIWKEACLART 3948 EGC922 INEPYCPFQNPFWYAECLART 3949 EGC923 TREPYCPFLNPTWLDQCIART 3950 EGC924 IREPYCPFLNPSWKAECIARS 3951 EGC925 ASEPYCPFINPTWKDECIARS 3952 EGC926 HSEPYCPFLNPTWKEQCTARS 3953 EGC927 MKEPYCPFLNPTWKAQCIART 3954 EGC928 TDETYCPFKNPIWYEECIARS 3955 EGC929 RGEPYCPFKNPTWKYQCLART 3956 EGC930 IREPYCPFLNPIWKAHCIALT 3957 EGC931 VSEPYCPFMNPIWKAECLSRT 3958 EGC932 EREPYCPFLNPIWLEQCLSRT 3959 EGC933 YSEPYCPFLNPIWKEQCIART 3960 EGC934 FGEPYCPFLNPTWKAECLVRS 3961 EGC935 LREPYCPFLNPTWKEQCLVRT 3962 EGC936 STDPYCPFLNPVWNYQCIART 3963 EGC937 SHEPYCPFQNPIWKAECLART 3964 EGC938 LNDPYCPFLNPIWKEQCMSRY 3965 EGC939 GYAPYCPFLNPIWKNECITRT 3966 EGC940 FSEPYCPFVNPYWKEQCIART 3967 EGC941 HTEPYCPFLNPTWKEQCLARF 3968 EGC942 GYEPYCPFLNPIWKAECKARS 3969 EGC944 LREAYCPFINPIWKDQCMARI 3970 EGC945 LIEPYCPFLNPHWKAECIARY 3971 EGC946 SSEPYCPFINPMWKDQCLART 3972 EGC947 PSDPYCPFLNPSWKAECLART 3973 EGC948 SSEPYCPFLNPIWKAECIARA 3974 EGC949 FPEPYCPFLNPHWKAQCLERL 3975 EGC950 FREPYCPFLNPTWKQQCLARI 3976 EGC951 PREPYCPFLNPIWKDECIART 3977 EGC952 MSDPYCPFLNPTWKAQCIART 3978 EGC954 NSEPYCPFSNPTWKDECLIRS 3979 EGC955 LREPYCPFLNPTWKEQCIARY 3980 EGC956 HNKTYCPFLNPTWKAQCIART 3981 EGC957 IGEPYCPFLNPTWKEECIRRT 3982 EGC958 ISEPYCPFINPTWKAECIARS 3983 EGC959 TKAPYCPFKNPIWKAECIART 3984 EGC960 PGDPYCPFLNPIWKQECIVRN 3985 EGC961 STEPYCPFSNPTWKAQCIARI 3986 EGC962 RSEPYCPFLNPTWKDQCIARS 3987 EGC963 SSEPYCPFLNPIWKEHCIART 3988 EGC964 YREPYCPFLNPIWKEQCIART 3989 EGC965 HSEPYCPFVNPTWYNECISRT 3990 EGC966 RSEPYCPFLNPIWKDQCIARS 3991 EGC967 VNEPYCPFLNPKWYAECLART 3992 EGC968 QNDPYCPFLNPIWKAQCIARS 3993 EGC969 SSEPYCPFLNPYWKEQCIART 3994 EGC970 YREPYCPFLNPYWKDQCMART 3995 EGC972 MHEPYCPFINPTWYEQCLARS 3996 EGC973 QLKPYCPFLNPIWYDQCVSRT 3997 EGC974 SSEPYCPFLNPSWKDQCIARL 3998 EGC975 YIDPYCPFLNPTWKDACLARS 3999 EGC976 SSEIIYCPFVNPAWKEACIARY 4000 EGC977 QSEPYCPFVNPTWKNECIARY 4001 EGC978 LSDPYCPFLNPIWKDQCIART 4002 EGC979 LGEPYCPFINPTWKAECLART 4003 EGC980 YGDPYCPFLNPTWKENCTART 4004 EGC981 LTEPYCPFLNPTWKDECLART 4005 EGC982 FREPYCPFKNPTWLAQCIARS 4006 EGC983 ANEPYCPFLNPHWKGQCVLRS 4007 EGC984 LNEPYCPFNNPTWKEECIKRS 4008 EGC985 QSEPYCPFLNPTWYTECVART 4009 EGC986 SSQPYCPFLNPTWKDACLART 4010 EGC988 SHEPYCPFLNPEWKAECIARN 4011 EGC989 ASDPYCPFLNPIWKDQCILRS 4012 EGC990 DNDPYCPFLNPTWKEECVARS 4013 EGC991 ESEPYCPFVNPIWKDQCMARY 4014 EGC992 LSAPYCPFLNPTWKDQCLART 4015 EGC993 SSEPYCPFLNPIWKQACYDRT 4016 EGC994 VDEPYCPFLNPNWKEACIARS 4017 EGC995 LIEPYCPFLNPTWKEQCFVRS 4018 EGC996 NDEPYCPFLNPIWKAQCLARN 4019 EGC997 AGKPYCPFLNPSWYDECIRRL 4020 EGC998 LSEPYCPFLNPIWKDQCMIRS 4021 EGC999 STEPYCPFLNPTWKDQCIARI 4022 EGC1000 PREPYCPFLNPVWKAQCIARW 4023 EGC1001 ISAPYCPFKNPIWKAECIARS 4024 EGC1003 IGEPYCPFLNPTWKDQCLVRS 4025 EGC1004 SSVPYCPFKNPTWKAECIART 4026 EGC1005 YREPYCPFLNPLWKDECIART 4027 EGC1006 HSEPYCPFLNPTWKDECITRT 4028 EGC1007 YSDPYCPFLNPSWYDECVSRM 4029 EGC1008 YREPYCPFLNPTWYVECIARS 4030 EGC1009 HREPYCPFLNPAWKEQCLVRS 4031 EGC1010 ASEPYCPFLNPTWRDACLART 4032 EGC1011 LREPYCPFLNPTWKEQCIARS 4033 EGC1012 SSEPYCPFLNPTWYNECIYRI 4034 EGC1013 LREPYCPFENPIWKEKCIAIT 4035 EGC1014 PSEPYCPFLNPTWKAECIART 4036 EGC1015 ATEPYCPFLNPIWKAQCIERM 4037 EGC1016 LGEPYCPFLNPTWKLQCLART 4038 EGC1017 ATEPYCPFLNPVWKDQCILRT 4039 EGC1018 PIEPYCPFINPMWKAECIART 4040 EGC1019 GREPYCPFLNPVWYNECMVRF 4041 EGC1020 VIEPYCPFLNPIWKEQCIARS 4042 EGC1021 TIEPYCPFLNPTWYKECLLRS 4043 EGC1022 LGEPYCPFLNPTWKDQCTARS 4044 EGC1024 LNEPYCPFLNPTWKAQCIART 4045 EGC1025 WSEPYCPFLNPTWKAECIART 4046 EGC1026 ASEPYCPFWNPIWKAQCIARI 4047 EGC1027 ISEPYCPFVNPTWKAQCIART 4048 EGC1028 VIEPYCPFVNPTWKEQCILRS 4049 EGC1029 MSCDEAWYNEQTWLWCVT 4050 EGC1030 WQCEWQWVQEWGAWDEFCVM 4051 EGC1031 GYCATDYWDYNGVWGTYCTQ 4052 EGC1032 RYCYLFEKYAPFYDQCLL 4053 EGC1033 RYCYLFQKYAPFYDQCLL 4054 EGC1034 GCCYWWGLVYDKWIKCET 4055 EGC1035 YQVKCPFQNPVWWSHCMYLL 4056 EGC1036 GYCFLFYKFPQYHMQCII 4057 EGC1037 KECLFTVPGVQHPVMWYCLT 4058 EGC1039 LLCRGQTANNLQPDECLT 4059 EGC1040 TCCRDYWMYRFLYTQCGM 4060 EGC1041 SFHRCFEWVRHNCVWGA 4061 EGC1042 ETLACAKLMPWYNECLARF 4062 EGC1043 RYCDHFVNHHDYYKLCLT 4063 EGC1044 WQCEWQWVQEWGAWDKFC VM 4064 EGC1045 LERLCIEIWSYHCRSFL 4065 EGC1046 LATWCQRWLASACFGLF 4066 EGC1047 GWCFEYYPEVSRFVYSDCNS 4067 EGC1049 YQVKCPFQNPVWWTHCMYLL 4068 EGC1050 WHCQWFWVPEWGAWDEFCVM 4069 EGC1051 STCLLQYYQFDVFSKKFCPL 4070 EGC1052 WHCEWHWVQEWGAWDEFCVM 4071 EGC1053 PKVRCPFQNPIWWSHCMEIL 4072 EGC1056 SVCHKMWPIIDDLYYCLR 4073 EGC1057 SLCQHFQQQPVWYQACIR 4074 EGC1058 YQVRCPFQNPVWWSHCMYLL 4075 EGC1059 GYCFLFYKFPQYHMQCIS 4076 EGC1060 LLCRGQSANNLQRDECLR 4077 EGC1063 WHCQWFWVHEWGAWNEFCVM 4078 EGC1066 WQCEWEWVQEWGAWDEFCVM 4079 EGC1070 DQVKCPFQNPVWWSHCMYLL 4080 EGC1072 WHCEWQWVQEWGAWDEFCVM 4081 EGC1073 PKVRCPFQNPIWWSIICMKIL 4082 EGC1074 GRCRGYEVDPWWHAPCFCDL 4083 EGC1075 ILCQHFQQQPVWYQACIR 4084 EGC1082 WNEPYCPFLNPTWKAQCIARI 4085 EGC1083 LGEPYCPFLNPFWYDQCIARN 4086 EGC1084 LGEPYCPFLNPYWYDQCMSRT 4087 EGC1085 ASSETYCPFLNPYWYDQCMSR TSGGGSGGGSQAAAGGSYMH EPHMQVLEIMN

TABLE 20 ErbB4 binding MRDs SEQ ID: Ref. No. Peptide Sequence 4095 EGD1 HPWRCLKKPEVVQCLRTG 4096 EGD2 GIFACVKEPEVVKCLISW 4097 EGD3 HPWRCLEEPEVVQCLRTG 4098 EGD4 EERLCYTPEAIFVDCTLHF 4099 EGD5 SRADWCDWHKCVVLQW 4100 EGD6 AYADWCSWHQCVQLDS 4101 EGD7 SVLVCWMYDQCVSVS 4102 EGD8 IIRWRCLIEPEVVQCLRTG 4103 EGD9 ADGHCWMYYEDWMCLYLM 4104 EGD10 SGWHCWVDYFCTRVE 4105 EGD11 VGAFCLPFEYWYQDCTRRL 4106 EGD12 PVETCMNYYEDIICRFLV 4107 EGD13 WKDFCPDVPPWLACTGSS 4108 EGD14 FPFSCVMRPHVIECLLSG 4109 EGD15 ADGHCWMYYQDWMCLYLM 4110 EGD16 EQLNCQEKIQYWCTRIY 4111 EGD17 AYADWCSCINASSWT 4112 EGD18 AYTDWCSWHRCVQLDS 4113 EGD19 YLIMCKEFPWSFTCGTLY 4114 EGD20 DNSGCNAPTFVCGFLW 4115 EGD21 SPFLCVHDFQVIECLIRW 4116 EGD22 QDNCCFIIIDNCQSGF 4117 EGD23 SVGRCFSHMPYHVSCSVSV 4118 EGD24 GWPPCADWMDQQLCSSAL 4119 EGD25 LKYRCVIYFHCYGWM 4120 EGD26 SQNVCLHLPWGWECFAWP 4121 EGD27 KPWRTCFNHGCELSLR 4122 EGD28 SRADWCDWXNASSCS 4123 EGD29 EGPPCYDWWTSSCARVR 4124 EGD30 ADGHCWMYYEYWMCLYLM

TABLE 21 DR5binding MRDs SEQ ID: Ref. No. Peptide Sequence 4125 DR51 VSDTCMVIMTCRYSP 4126 DR52 SERICFKIYMTSKNTCQKAS 4127 DR54 ALRPCLVYMSMKCSVTW 4128 DR55 SLTPCVYLTMCAPGH 4129 DR57 DVGECRVIMTCRPSS 4130 DR58 QQGSCKIYMTCRYSH 4131 DR59 QWQVCYWTEGRWADSCGWAQ 4132 DR510 SVYPCKVIMTCLRSP 4133 DR512 TGNTCKRPVLTCNWSG 4134 DR513 TEFGCQVYMTCRPKL 4135 DR514 SVYPCKVIMTCLKSP 4136 DR515 NFRMCIPIMTMHCWADD 4137 DR516 DQYTCDRVILTCWTPH 4138 DR517 RVSECFLYLTCHKGQ 4139 DR518 PTRECTVILTQHCWNVW 4140 DR519 LTPCQPIMTCKRPV 4141 DR520 SPSPCKVIMTCAPLN 4142 DR521 ARHPHEPPYLCMKCL 4143 DR523 ANDRCIPIMHWHCWAYW 4144 DR524 VQGGCDLVILTCKMWG 4145 DR525 LRDFCSTTMAHQCHFGP 4146 DR526 AKGECQVYLTCRYNS 4147 DR527 GSEGCKVILTMKCPDRI 4148 DR528 EYVECGPHQYYWFGHCHFWA 4149 DR529 KCNFNPWKAACG 4150 DR530 YAERCKVYLTMKCDISQ 4151 DR531 PSEMCVVVMRNYCWSST 4152 DR533 LSSGCMVYMTMKCGSTK 4153 DR534 VGADCWVIMTCRKNL 4154 DR535 ATTPCMVYMTCFRTP 4155 DR537 QDRACFPIMTLHCPGEG 4156 DR539 QTRNCGVWPNHLVCMPLS 4157 DR540 LQKVCAAWYYCMESH 4158 DR541 LRANCKVVLTERCGNYL 4159 DR542 ADEACLVVLTCRASP 4160 DR543 SGLGCPQNHLLVLTCRHAM 4161 DR544 WIAGCKLVLTERCLNYF 4162 DR545 NNSQCQVILTHRCHSLT 4163 DR548 KVSVCFPILTMYCSERY 4164 DR550 NDLMCQPIMTMKCARYQ 4165 DR551 ILIFCQTPPHHCLSKL 4166 DR552 SYQRCKVIMTQKCIVPP 4167 DR553 TSDRCVVVLTCRMWG 4168 DR554 HVSRCEPVLTMHCWRGS 4169 DR555 PSYPCSIILTCRSTL 4170 DR557 RDSECFIYLTCRVLP 4171 DR558 SAEDCFVILTCLCSR 4172 DR559 DRYPCYFYLTCIPAS 4173 DR561 LQSECFVIMTCKRVN 4174 DR562 NTKPCMPIMTCLKWN 4175 DR563 WNTPCMVIMTCRPHE 4176 DR564 PLTKCKVIMTCARGI 4177 DR565 WHYPCQPVLTCRSHN 4178 DR566 FQTECLVYMTCRTIY 4179 DR568 WESPCFIKLTCHMVA 4180 DR569 FVGHCWWPLGLCAPLP 4181 DR570 SSMSCVEYQQCRLGY 4182 DR571 ARDSCLVYMTCRPVA 4183 DR573 LNASCMVYLTQRCSHTS 4184 DR574 RIIDGCNKIMLTCIITRA 4185 DR575 HRDPCTVVETCRSLS 4186 DR576 MTSECQKVILTCWNAS 4187 DR577 YMSVCHIDGHQVVCLKNK 4188 DR580 SGDTCLVILTHRCATVS 4189 DR581 YTAPCKVYMTCVRPM 4190 DR582 LRANCKVVLTERCCNYL 4191 DR583 PPSSCQVILTCRPIW 4192 DR584 LSTRCTDQDIVLTCRIKD 4193 DR586 WHYPCQPVLTCRSRT 4194 DR588 GQTDCMIIMTCKQWK 4195 DR589 PATRCEPVLRHFCWGQG 4196 DR590 DSFGCTWEVWGRECHPQL 4197 DR591 FYLYCPYPEMSSHCVLRA 4198 DR592 LRADCKVVLTERCSNYL 4199 DR595 HENPCQVYMTCRHLD 4200 DR596 SAEECFVILTCLGSR 4201 DR597 ARQDCVPVMRWHCWAEI 4202 DR598 PPLGCTRIALTCWDIR 4203 DR599 QPPECRVIMTLRCGDAW 4204 DR5100 PKQACKIIMTERCWAVD 4205 DR5101 TVGECMPIMTCFSTN 4206 DR5102 WHYPCQPVLTCRSHT 4207 DR5104 PLTKCKVIMTCAWGI 4208 DR5105 GISGCKLVLTQRCLHVN 4209 DR5107 ERQGCVVYLTERCVVGR 4210 DR5109 NNSQCQVILTHHCHSLT 4211 DR5110 DKSPCQVVMTCRPLL 4212 DR5111 ALHKCMVILTNRCETPV 4213 DR5112 TTTSCKLYLTSTCSPNH 4214 DR5113 TEYPCKVYMTCMWPI 4215 DR5114 LGPNCKIVLTERCGNSL 4216 DR5115 SERPCGERPVVMTCFPVR 4217 DR5116 GSFSMRWDCGDNPIPGRRQCVIM 4218 DR5117 GSAQLLWDCGDNPIMGRRQCVKL 4219 DR5118 GSYTHTFDCGDFNITGRRQCVKL 4220 DR5119 GSLSSQFDCGENPILGRRQCLYL 4221 DR5120 GSRAQIWDCGDNPIRGQRQCVKL 4222 DR5121 GSIHIPPRCTEEDVIMTCRRSAD 4223 DR5122 GSSTFPRFCAEEDVIMTCRWRFD 4224 DR5123 GSNAIPRHCTEEDVIMTCRLRPS 4225 DR5124 GSAEFHRPCGEEDVIMTCRWRVH 4226 DR5125 GSNALPRACDERDVIMTCRKSVA 4227 DR5126 GSYDFPRTCAEEDVIMTCKWRIS 4228 DR5127 GSIPSRFDCGENPIAGRRQCVEL 4229 DR5128 GSQYIHMDCFENRIRGYCHCIKM 4230 DR5129 GSILIPRTCAQEDVIMTCRERAY 4231 DR5130 GSQGLPRPCGEEDVIMTCRWSGY 4232 DR5131 GSDSFTRTCTEDAVTMTCKWRVL 4233 DR5132 GSDPSPRTCAEEDVIMTCRWQVY 4234 DR5133 GSSMITRPCAEEDVIMTCKWLPI 4235 DR5134 GSGYNPRLCSEQDVIMTCRLRFG 4236 DR5135 GSFDRNWDCGDNPIKGRRQCVKV 4237 DR5136 GSTYMQFDCGDNPIPGRRQCVPL 4238 DR5137 GSFVSHWDCGENPIIGRRQCVLL 4239 DR5138 GSPPHSRPCAEEDVIMTCRYRIY 4240 DR5139 GSLDRPRPCAEEDVIMTCRWRAA 4241 DR5140 GSWKWSPTCAEEDVIMTCRKHVK 4242 DR5141 GSKSVPRLCSEEDVIMTCKRGVF 4243 DR5142 GSEYWQPPCAEEDVIMTCKWRVV 4244 DR5143 GSLCLPRHCAENEVIMTCRPLMF 4245 DR5144 GSLMSQWDCGDNPILGRRQCVKL 4246 DR5145 GSFKTIWDCGDNPIPGRRMCVKL 4247 DR5146 GSSSITWDCGDNPIPGRRQCVIII 4248 DR5147 GSPRFKWDCGDNPIAGRRQCVLL 4249 DR5148 GSSGFPHHCGEEDVIMTCRWRGL 4250 DR5149 GSPHFPRPCAEEDVIMTCRWRVI 4251 DR5150 GSGWHTRFCAEEDVIMTCKWRVI 4252 DR5151 GSDHWRRSCSEDDVIMTCRWRVS 4253 DR5152 GSFKVPHLCAEEDVIMTCRWRVY 4254 DR5153 GSRSSPHPCGEEDVIMTCRWRIT 4255 DR5154 GSVLQVCDCLDIPVFGTCQCVQL 4256 DR5155 GSGDSHWDCGDNPILGRRQCVKL 4257 DR5156 GSTSMLWDCGANPIKGRRQCVLL 4258 DR5157 GSMPYSWDCGDNPIVGRRQCVKV 4259 DR5158 GSFTSRWDCGNNLVIGQRQCIKL 4260 DR5159 GSSAPGFDCGDNPIVGQRQCIKL 4261 DR5160 GSMVGQRPCAEEDVIMTCRWRVV 4262 DR5161 GSSLLPRNCSEEDVIMTCRWRVD 4263 DR5162 GSGTTPRRCAEEDVIMTCRWSAH 4264 DR5163 GSSYSPRPCAEEDVIMTCRPRFH 4265 DR5164 GSGVFPRPCHEEDVIMTCRWRVH 4266 DR5165 GSADPLWDCGDNPIKGRRQCVPL 4267 DR5166 GSLPHAWDCGDNPIPGRRQCVKL 4268 DR5167 GSTKLTHPCGEEDVIMTCRWRVA 4269 DR5168 GSTASPRHCAEHDVIMTCRWPVD 4270 DR5169 GSERLARSCDEDDVIMTCRWRVT 4271 DR5170 GSNKPPRPCSEEDVIMTCRWRVV 4272 DR5171 QGSDEWDCLDNRIGRRQCVKL 4273 DR5172 GSSLSSWDCGDNPIPGRRQCVKL 4274 DR5173 GSPSEYWDCGDNPIFGRRQCVRL 4275 DR5174 GSLSPLWDCGDNPILGRRQCVLL 4276 DR5175 GSAAHYWDCGDNPIVGRRQCVKL 4277 DR5176 GSYLYPRPCSEEDVIMTCRWRVS 4278 DR5177 GSSSFTPTCNEEDVIMTCRWRVV 4279 DR5178 GSLSAPPTCAEEDVIMTCRWRLK 4280 DR5179 GSRNFPVPCDESDVIMTCKWRVS 4281 DR5180 GSDRFQRHCSEADVIMTCKSRLY 4282 DR5181 QGSDEWDCLDNRIGKRQCVKL 4283 DR5183 GSSSFPRPCAEEDVIMTCKWRVY 4284 DR5184 GSMLLPRSCAEEDVIMTCRWAHY 4285 DR5185 GSGKCTEEDVIMTCRSKVT 4286 DR5186 GSSHFPHPCSEEDVIMTCRWRVN 4287 DR5187 GSVDFPRPCLEDDVIMTCKWRLY 4288 DR5189 GSVLDILSCKCLDTSSHVQCVTL 4289 DR5190 GSRMSSPSFDCKGDFGHRQCVKL 4290 DR5191 GSGEPSDGRVCLTKCSSLIQCFKF 4291 DR5192 GSHCLYVVWECVDHGICLRRCAEL 4292 DR5193 GSNNTTSMWDCLDRPIGQRQCVKL 4293 DR5194 GSYWSFSKWDCLDRPLGQRQCVLL 4294 DR5195 GSPTVPNQCHCQDNRISRMCVIL 4295 DR5196 GSDKFRERWDCLDYRIGHRQCIKL 4296 DR5197 GSMYTDDDRVCLTTCSVLRQCIKL 4297 DR5198 GSDLFSSRWDCLGKLGQRQCVKL 4298 DR5199 GSQLLTFGWDCLDRPIGQRQCVKI 4299 DR5200 GSGDEHAYWDCLDRPIGQRQCVKM 4300 DR5201 GSSGFYERWDCLDHHIGRRQCVKL 4301 DR5203 GSMKVTGPWDCLDRPIGQRQCVYL 4302 DR5204 GSLPDTYLTNCIHNRHDLCLCSKII 4303 DR5205 GSRSSSMPSVCLTDCISLRQCVKL 4304 DR5206 GSSNSMEPRVCLTNCTALRMCAKL 4305 DR5207 GSRIDLSQWDCLDYRLGQRQCVKV 4306 DR5209 GSIGHSPRWDCVGKPGHRQCVKL 4307 DR5210 GSQLLTSMFDCGDRPMGRRQCVLV 4308 DR5211 GSPHPTATRVCLTDCLSLRKCLKM 4309 DR5212 GSCTGVLTQACPAQNSFLQCVEL 4310 DR5213 GSSNFESRWDCIGNVGSRQCVKL 4311 DR5214 GSHNHYDDQVCLTQCNNLRLCIEL 4312 DR5215 GSQAPNIESLCLTRCVNLLHCIKI 4313 DR5216 WDCLDRPLGQRQCVLL 4314 DR5217 FDCKGDFGHRQCVKL 4315 DR5219 WDCLDRPIGQRQCVKM 4316 DR5220 WDCLDYRLGQRQCVKV 4317 DR5221 WDCLDRPIGQRQCVYL 4318 DR5222 WDCVGKPGHRQCVKL 4319 DR5223 WDCLDYRIGHRQCIKL 4320 DR5224 WDCIGNVGSRQCVKL

TABLE 22 CD3 binding MRDs SEQ ID: Ref. No. Peptide Sequence 4329 CD32 FDLLCVHVESHKYYCFRPI 4330 CD34 FILSCHWHNGVVRCYEVS 4331 CD38 PPFSCVFDVPTWSWVCGQKS 4332 CD311 AQLLCIIIIVTGKYFSCREVI 4333 CD317 DWWLCESEKQTFWCSILR 4334 CD318 RGMQCHFEKTGWMWVCSLAG 4335 CD323 FMMQCQYVMNGIYCYDSG 4336 CD324 FRLGCVPLPVIQACVAQAAAG GSKIWECEQNPQVWICNLTS 4337 CD326 PKLLCHDTEDIYCFCELLK 4338 CD328 LFGRCYLYHNLAWYHCVLVW 4339 CD329 IGLLCKKDHGEWSCFRIY 4340 CD330 FMVDCQKMPHYIICAERG 4341 CD331 EWQCAYYPQYFQWQCEILG 4342 CD332 GPPWCMWHQRQYSWICIFGE 4343 CD335 IAWDCQWHHQWHAWECTMNS 4344 CD337 WDAVLCVNKRCSGLYF 4345 CD338 TVVRCYQSPYVYWICKFPT 4346 CD340 FSVICWQKPDYLYCAEDA 4347 CD341 RTVYCGWDANRQAWLCHCQN 4348 CD342 PGFRCSWWEIAKSWVCTMPV 4349 CD343 DLLDCSFNEFEWHWTCSFTQ 4350 CD345 PLVSCQYFPPLHHWRCDFLV 4351 CD347 DKWKCRWKEAYICMRDS 4352 CD348 YRLSCQYLSAHQVICSGLW 4353 CD349 RWAYCSVQDIMWDCTLFP 4354 CD354 TMITCHWRKDHYDWMCQLEY 4355 CD356 FILSCHWNLGVVPCYEVS 4356 CD357 PVMSCEPIPIGWKCSLQT 4357 CD360 FSPECHYFPGYWKCTWSL 4358 CD361 WPQRCVWWWEMDYKCRSLI 4359 CD365 TYWDCVVGHKHMQCLRLS 4360 CD367 SRGWCQYEYQHSQWRCFIHE 4361 CD368 RLVSCSWENSKWQWTCSVLE 4362 CD369 PDSSCHTLYWQLHHCARRE 4363 CD370 GQVTCNYNPATHSWVCSCIS 4364 CD3309 TVFRCVFVNEYRVWFCHIST 4365 CD3318 GEEVCYWAPFEPVRCLPVW 4366 CD3323 VFMVCVHHYEKVKCWAFW 4367 CD3324 FFALCKFQNFHTATCLKLI 4368 CD3354 WSFLCIKVTQQHYECFLFS 4369 CD3364 GLVSCVILPQGWFCFKTA 4370 CD3366 QVKYCVVTHMMCVIDL 4371 CD3399 RSALCHDKMTVKSYCLSSR 4372 CD3412 IFFVCPNNQYDCAVNH 4373 CD3430 WNFFCITFGPYLHCWIYT 4374 CD3431 TMEVCYWGPFTTATCERWP 4375 CD3432 WECYCQGIIVIIYEYCVCFR 4376 CD3433 NHWFCFTDWHHFVCFKFA 4377 CD3435 CWVSCWIWPNMWYCWLDC 4378 CD3436 GVGGLLLGSVRTGSLPPGVGPG 4379 CD3438 YAAWCFKLNKLEVTCFSGI 4380 CD3439 ARFVCFSLVEAQWYCLVLS 4381 CD3440 IYFTCVWYAQQFWCIVMP 4382 CD3444 RGWVCYFFPPHFVCYTWV 4383 CD3446 FSLVCIQMYNQVYCVFIL 4384 CD3447 QDFLCVKIEPFPWICWTMQ 4385 CD3448 FTCVCYQHYDHFWCGCLW 4386 CD3449 ILWACVKQYDGMICWSPS 4387 CD3450 FWLLCIHAPKQDVCFIWM 4388 CD3453 FRALCWEHPPTRYCILFT 4389 CD3454 TAWWCVIMPPKFFCASFN 4390 CD3455 GRVSCYWETHLNVVCVPSL 4391 CD3457 IKLECKVIIAPSMLSCTLWQ 4392 CD3458 YEWSCLITPYGHWCAQTV 4393 CD3459 FTLLCIKFELKPYICWVSL 4394 CD3461 TPRGCTFIFIMPDMCRPSL 4395 CD3462 LRCTCWIFPKNVYCLCLS 4396 CD3463 AFLLRCYAQECVILWL 4397 CD3465 GMVSCVIMPQGWFCFKTA 4398 CD3466 MHWVCIIFPPGFFCVSTF 4399 CD3470 LHFRCFQLYNEYWVCIMSV 4400 CD3474 WNFLCFFTHHKPVWYCVWTM 4401 CD3475 GFQCWCPHVVCFRVPY 4402 CD3477 MQFLCLTVQTNTWYCFLWL 4403 CD3478 FIVRCVIGQKLVCVQTE 4404 CD3479 WSPNCLWFIKTTYVVCPDST 4405 CD3480 YAWECQWIVKPHQFVCGGM 4406 CD3481 GTLRCYSLNWGYFCILFV 4407 CD3482 NKVECHIVLWTFHFGCPSAT 4408 CD3483 TSMVCRSMHMYWLCVNLD 4409 CD3487 NRVLCMMNYIHVTKLCDRPM 4410 CD3490 PLMLCRHMKHFQYYCWPLA 4411 CD3491 PSQRCVNHFVTFFNCTQSN 4412 CD3492 WNFFCITFSPTCIVDLYWPG 4413 CD3493 WTMRCYSHAPNKLVCSVGL 4414 CD3494 LVLHCDYQQGVWTCKAVL 4415 CD3495 VMFVCFSLPEQQVCVPLS 4416 CD3689 PVQICQWTLELQCSPWT 4417 CD3690 LQWVCHWVAECSMSP 4418 CD3693 RGFICEWNLVASCVPAT 4419 CD3694 RIWICDWTIECVESL 4420 CD3697 FVCECFAWDWFGPWQCMCSV 4421 CD3700 RFWIECWTEACRPGLLGQAGQ GGPGSAGGSYPSIECYSYMCN VLWF 4422 CD3703 LRSSCEYHQTCNQSSQAAAGG SREMKCDWRVDCLYYV 4423 CD3704 TLTVCQWVLHATCVDVQ 4424 CD3706 VSIRCHQYLLCVFES 4425 CD3707 SAWFCEWTFQCQLMG 4426 CD3708 GFIVCYWEAVCQSST 4427 CD3709 VLWRCTWVMECVSVS 4428 CD3718 DMWLCHWRIECVPYL 4429 CD3724 PWASCYFYQEHVHCEPPD 4430 CD3743 SYSVCEWEFVYSCWPPY 4431 CD3789 RQQPCLKIWSCLQLY 4432 CD3793 PVQICQWTLELPCSPWT 4433 CD3832 PVPICPWTLELHCPPWTGHA 4434 CD3872 MACLCVSWSKCTPLG 4435 CD3882 QTMKCPMEMGHCTTWR 4436 CD3912 FILLCIIQYLVCIKQE 4437 CD3919 FYMRDCESHRCRLDLS 4438 CD3924 GFQLACRFMSCGCRSC 4439 CD3935 PLMLCRHMKHFEYYCWPL 4440 CD3940 QDFLCVKIEPFPWICWTMQGQ 4441 CD3942 IYFYCVEWYQKWICYTVV 4442 CD3952 VTCFYVETDHVVCFREA 4443 CD3959 GRVSCYWETHLNVVCVPSL 4444 CD3960 FPCVCIMTHHHLECFCFIGQ 4445 CD3972 FSLVCIQMYNQVYCVFILGQ 4446 CD3981 ILWACVKQYDGMICWSPSGQ 4447 CD3992 VKLPCFLIQEMGWMCFWPL 4448 CD3998 WNFFCINFSPYLHCWIYTGQ 4449 CD31008 MYVLCKEAQVGQQWKCVQFF 4450 CD31014 YTPECGWGYMIMFKHCRHYE 4451 CD31025 FWLLCLHVWNKTTVYCLVKH 4452 CD31032 FQCVCVHWNDGRVVCLCHY 4453 CD31034 DYTSCQFRGWLICEAMA 4454 CD31035 LVMLCWKWHEVRHCLLLS 4455 CD31039 VVLTCSSSWERSVFVCRLVE 4456 CD31045 LAFLCIHWQEHLWCIVRP 4457 CD31047 SVVTCYIIMFPWDRIICFLQ 4458 CD31051 TVRCWCEHMVCFMERD 4459 CD31054 CHFLCFKLGFMEYFYCEYSC 4460 CD31058 VMLACYKHPLNNSEWCYILP 4461 CD31063 FRVLCADVPGHRYCFIVS 4462 CD31068 LVFRCLHITQHWFCFGLT 4463 CD31077 EYDSCMFYGFIICPKQT 4464 CD31079 MLFVRCEHGRCTVYEI 4465 CD31086 CEIFCMVSLETQKMVCVIIRA 4466 CD31120 MGFLCWSQPGNEYEWCVPIN 4467 CD31138 RFIHCVHFEWINHIVCFVGT 4468 CD31162 QDFLCVKIEPFPWICWTMQ 4469 CD31165 DRALCWLHPHFATVQCIIMS 4470 CD31173 VTCFYVETDHVVCFREA 4471 CD31179 GRVSCYWETHLNVVCVPSL 4472 CD31180 FPCVCIMTHHHLECFCFI 4473 CD31190 FSLVCIQMYNQVYCVFIL 4474 CD31199 ILWACVKQYDGMICWSPS 4475 CD31211 ADSWCHTLYWNLRHCEIQE 4476 CD31217 WNFFCINFSPYLIICWIYT 4477 CD31249 ESLDCATPLLLCTGCGDQA 4478 CD31254 SFQMCMGWFWWCWSHS 4479 CD31255 WSQSGCTYAHCMYCIH 4480 CD31420 VNCWLHLQFFYSEQYCIE 4481 CD31567 ATGGSLVVKCDWWLDCLVYL 4482 CD31579 DLQICHWTLELECSPRK 4483 CD31605 ATGGSREIRCKWRVDCMCYS 4484 CD31631 SLSSCELYYKCRHFSQAGQGS ATGGSLVVKCDWWLDCLVYL 4485 CD31644 LRCFCDYYQTCKQYTVAGQGS ATGGSREIRCKWRVDCMCYS 4486 CD31656 WNYFCVYFSPYLIICWIYS 4487 CD31663 WNFVCIFVSPYMHCWLYF 4488 CD31697 WKFLCESTLSPYLHCWIYP 4489 CD31718 WNFFCIYISPHVHCWIHS 4490 CD31727 TAWDCVVGQKYMLCQRLS 4491 CD31803 CQERCGKMIKSIVMQCNLRL 4492 CD31909 SVSVCVEHAYGQWYCFAAR 4493 CD31915 TMKCPMEMGHCTTWR

Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims

All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference. 

What is claimed is:
 1. A modular recognition domain (MRD) capable of binding an epidermal growth factor receptor (EGFR), wherein the MRD comprises the amino acid sequence YAEHCRQFPSDWICTLLP (SEQ ID NO: 3109).
 2. A complex comprising the MRD of claim 1, wherein the MRD is fused to a heterologous protein.
 3. The complex of claim 2, wherein the heterologous protein is an antibody.
 4. The complex of claim 3, wherein the MRD and the antibody bind different targets.
 5. The complex of claim 3, wherein the MRD and the antibody are operably linked by a linker.
 6. The complex of claim 5, wherein the antibody binds EGFR.
 7. The complex of claim 6, which further comprises an additional covalently bound MRD, wherein the additional covalently bound MRD binds Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3) and comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3499-4094.
 8. A pharmaceutical composition comprising the complex of claim
 6. 9. The complex of claim 6, wherein the antibody is cetuximab.
 10. The complex of claim 9, which further comprises an additional covalently bound MRD, wherein the additional covalently bound MRD binds ErbB3 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3499-4094.
 11. A pharmaceutical composition comprising the complex of claim
 9. 12. A complex comprising an antibody and an MRD, wherein the antibody is cetuximab and the MRD comprises the amino acid sequence YAEHCRQFPSDWICTLLP (SEQ ID NO: 3109).
 13. A pharmaceutical composition comprising the complex of claim
 12. 